Observation method using binding and dissociation probe

ABSTRACT

An observation method of a sample containing a target substance, the observation method including an imaging step in which a step of obtaining a speckle image including, as a speckle, light emitted from a luminescent substance in which a medium is brought into contact with the sample is performed a plurality of times so as to obtain a plurality of speckle images, the medium containing a probe that contains the luminescent substance emitting light and that repeatedly binds to and dissociates from the target substance directly and specifically, and an observation image generation step of generating an observation image of the target substance in the sample from the plurality of speckle images, wherein a half-life of a probe-target complex formed by binding between the probe and the target substance is equal to or more than 10 milliseconds and equal to or less than 3 seconds.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is based upon and claims the benefit of priority from prior Japanese Patent Application No. 2015-048692, filed Mar. 11, 2015, the entire contents of which are incorporated herein by this reference. This is a Continuation Application of PCT Application No. PCT/JP2016/057817, filed Mar. 11, 2016, which was not published under PCT Article 21 (2) in English.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention is related to an observation method using a binding and dissociation probe by which a super-resolution image of a sample can be obtained.

Description of the Related Art

In recent years, super-resolution microscopy (STED, SIM, PALM/STORM; resolution capability:10 nm through 100 nm) with a resolution capability exceeding that of optical microscopes (>200 nm) has been developed (Nat. Rev. Mol. Cell Biol., 9, 929-943, 2008), and major microscope manufacturing companies (Nikon, Carl Zeiss, Leica) are selling their super-resolution microscope apparatuses. In the field of biological science particularly, important discoveries are expected from applications of PALM (photoactivated localization microscopy, Japanese National Publication of International Patent Application No. 2008-542826, Science, 313, 1642-1645, 2006)/STORM (stochastic optical reconstruction microscopy, Nature Methods, 3, 793-795 and United States Patent Application No. US2008/0032414). In each super-resolution microscopy, fluorescent dye is made to bind to a target so as to label the target, and the spatial distribution of the fluorescent dye is observed with a high resolution capability in Stimulated Emission Depletion (STED), structured illumination microscopy (SIM), and localization microscopy (PALM/STORM).

The outline of localization microscopy is as follows. First, a target substance to be observed is labeled with a luminescent substance such as a fluorescent substance. Then, the luminescent substance with which the target substance is labeled is made to emit light at a low density so that speckle images are obtained in which the speckles of emissions of light are separated individually, i.e., in which luminescent substances of single molecules are separated. The central positions of the individual speckles can be obtained in the respective speckle images that have been obtained. The above step of obtaining speckle images including separated speckles so as to obtain information of the positions of speckles is repeated a plurality of times, e.g., hundreds of times through hundreds of thousands of times, and the central positions of roughly all luminescent substances on the target substance are obtained, and thereby an observation image of the target substance is constructed. Explanations will be schematically given for the localization microscopy by referring to FIG. 1. For example, it is assumed as shown in FIG. 1 (a) that five molecules 100 of target substances to be observed are distributed. The five molecules 100 of the target substance are respectively labeled with luminescent substances and are made to emit light separately in such a manner that the speckles of light emission do not overlap each other. For example, as shown in FIG. 1 (b), three speckle images 110, 111 and 112 having separate speckles 101 are obtained at different times t1, t2 and t3. A gauss function is fit to the image of each speckle 101 in each of the speckle images 110, 111 and 112 so as to obtain the central positions of the speckles, and position information 102 which is information of the central positions is recorded (FIG. 1 (c)). Pieces of position information 102 of individual recorded speckles are synthesized so as to draw an observation image 120 as shown in FIG. 1 (d). The above method can be implemented by using for example DAOSTORM, a computer program (Nature methods, 8 279-280, 2011). In localization microscopy, as schematically shown in FIG. (1 b), it is necessary that images of the molecules 100, which are labeled target substances, be picked up over time while the molecules 100 are sequentially made to emit light at a relatively low density so as to obtain a plurality of speckle images so that the speckles of luminescent substances of single molecules can be separated.

PALM/STORM use a fluorescent substance that can perform activation or switching through irradiation with light for making a fluorescent substance emit light at a low density so as to adjust a light irradiation condition in order to stochastically perform activation or switching on the state of the fluorescent substance, and thereby obtain speckle images in which speckles are separated individually.

Proceedings of the national Academy of Sciences of the United States of America 103, 18911-18916 (2006) reports a method referred to as PAINT (point accumulation for imaging in nanoscale topography). The document discloses that super-resolution observation of a form of a lipid bilayer was conducted through PAINT by using the fluorescent dye Nile-red that swiftly goes back and forth between an aqueous solution and the lipid bilayer.

Recently, as a multicolor super-resolution microscopy to which PAINT above is applied, a method has been reported in which a target substance is labeled with an antibody that was made to fuse with a DNA oligomer and a fluorescent DNA oligomer which is a complementary base sequence that temporarily binds to and dissociates from the DNA oligomer is used (Exchange-PAINT, Nature methods, 11, 313-318, 2014).

SUMMARY OF THE INVENTION

The present inventors have found that a luminescent probe can be used for labeling different positions on a target substance in respective speckle images so that the labeling density with respect to the target substance can be substantially increased by increasing the number of the speckle images that are picked up and a high resolution observation image exceeding a diffraction limit can be generated by the localization microscopy from respective speckle images when a plurality of images of light emission speckles (speckle images) are picked up at different times and while keeping a contact state between the luminescent probe and a sample, by using the luminescent probe, as a binding and dissociation luminescent probe, that is a luminescent probe which repeatedly binds to and dissociates from a target substance directly and specifically and for which the half-life of a probe-target complex formed by binding between the luminescent probe and the target substance is in a prescribed scope, and the present inventors have completed the present invention. The present invention incorporates the following inventions.

(1) An observation method of a sample containing a target substance, the observation method comprising:

-   -   an imaging step in which a step of obtaining a speckle image         including, as a speckle, light emitted from a luminescent         substance under a prescribed condition in a state in which a         medium is brought into contact with the sample is performed a         plurality of times at different times respectively so as to         obtain a plurality of speckle images, the medium containing a         probe that contains the luminescent substance emitting light         under the prescribed condition and that repeatedly binds to and         dissociates from the target substance directly and specifically;         and     -   an observation image generation step of generating an         observation image of the target substance in the sample from the         plurality of speckle images, wherein     -   a half-life of a probe-target complex formed by binding between         the probe and the target substance is equal to or more than 10         milliseconds and equal to or less than 3 seconds.         (2) The method according to (1), wherein     -   the observation image generation step is a step in which         information of a position of a speckle included in each of the         plurality of speckle images is obtained for each of the         plurality of speckle images and the observation image is         generated on the basis of the information from the plurality of         speckle images.         (3) The method according to (1), wherein     -   the sample includes two or more target substances, the imaging         step is sequentially performed on the sample by using the probe         that is specific to each of the target substances, and     -   the observation image generation step is a step in which         observation images of the respective target substances in the         sample are respectively generated from the plurality of speckle         images obtained from the respective imaging steps.         (4) The method according to (3), further comprising     -   a multiple-observation image generation step in which         observation images of the respective target substances in the         sample generated in the observation image generation step are         superposed so as to generate a multiple-observation image, which         is an observation image of the two or more target substances in         the sample.         (5) The method according to (1), wherein     -   the luminescent substance is a fluorescent substance, and the         prescribed condition is irradiation with excitation light.         (6) The method according to (1), wherein     -   a combination between the probe and the target substance is         selected from a group of:         -   a combination wherein the probe is (a1) a polypeptide,             linked to the luminescent substance, which consists of an             amino acid sequence of sequence number 19, (a2) a             polypeptide, linked to the luminescent substance, which             consists of the amino acid sequence described in (a1) where             one or a plurality of amino acids have been substituted,             deleted, inserted or added and for which a half-life of a             probe-target complex formed by binding between the probe and             the target substance is equal to or more than 10             milliseconds and equal to or less than 3 seconds, or (a3) a             polypeptide, linked to the luminescent substance, which             consists of an amino acid sequence having at least a 70%             identity with the amino acid sequence described in (a1) and             for which a half-life of a probe-target complex formed by             binding between the probe and the target substance is equal             to or more than 10 milliseconds and equal to or less than 3             seconds, and the target substance is an actin polymer;         -   a combination wherein the probe is (b1) a polypeptide,             linked to the luminescent substance, which consists of an             amino acid sequence that is a partial amino acid sequence of             an amino acid sequence of sequence number 12, that at least             partially contains an amino acid sequence of 3-309 and that             has 407 or fewer amino acids, an amino acid sequence that is             a partial amino acid sequence of an amino acid sequence of             sequence number 14, that at least partially contains an             amino acid sequence of 2536-2843 and that has 408 or fewer             amino acids, an amino acid sequence that is a partial amino             acid sequence of an amino acid sequence of sequence number             14, that at least partially contains an amino acid sequence             of 2781-2819 and that has 138 or fewer amino acids, an amino             acid sequence that is a partial amino acid sequence of an             amino acid sequence of sequence number 4, that at least             partially contains an amino acid sequence of 1-908 and that             has 1008 or fewer amino acids, an amino acid sequence that             is a partial amino acid sequence of an amino acid sequence             of sequence number 4, that at least partially contains an             amino acid sequence of 659-908 and that has 394 or fewer             amino acids, an amino acid sequence of sequence number 5, or             an amino acid sequence of sequence number 6, (b2) a             polypeptide, linked to the luminescent substance, which             consists of the amino acid sequence described in (b1) where             one or a plurality of amino acids have been substituted,             deleted, inserted or added and for which a half-life of a             probe-target complex formed by binding between the probe and             the target substance is equal to or more than 10             milliseconds and equal to or less than 3 seconds, or (b3) a             polypeptide, linked to the luminescent substance, which             consists of an amino acid sequence having at least a 70%             identity with the amino acid sequence described in (b1) and             for which a half-life of a probe-target complex formed by             binding between the probe and the target substance is equal             to or more than 10 milliseconds and equal to or less than 3             seconds, and the target substance is a microtubule;         -   a combination wherein the probe is (c1) a polypeptide,             linked to the luminescent substance, which consists of an             amino acid sequence that is a partial amino acid sequence of             an amino acid sequence of sequence number 8, that at least             partially contains an amino acid sequence of 3777-4684 and             that has 1008 or fewer amino acids, an amino acid sequence             that is a partial amino acid sequence of an amino acid             sequence of sequence number 8, that at least partially             contains an amino acid sequence of 3777-4364 and that has             688 or fewer amino acids, an amino acid sequence that is a             partial amino acid sequence of an amino acid sequence of             sequence number 8, that at least partially contains an amino             acid sequence of 3777-4313 and that has 637 or fewer amino             acids, or an amino acid sequence that is a partial amino             acid sequence of an amino acid sequence of sequence number             8, that at least partially contains an amino acid sequence             of 4022-4364 and that has 443 or fewer amino acids, (c2) a             polypeptide, linked to the luminescent substance, which             consists of the amino acid sequence described in (c1) where             one or a plurality of amino acids have been substituted,             deleted, inserted or added and for which a half-life of a             probe-target complex formed by binding between the probe and             the target substance is equal to or more than 10             milliseconds and equal to or less than 3 seconds, or (c3) a             polypeptide, linked to the luminescent substance, which             consists of an amino acid sequence having at least a 70%             identity with the amino acid sequence described in (c1) and             for which a half-life of a probe-target complex formed by             binding between the probe and the target substance is equal             to or more than 10 milliseconds and equal to or less than 3             seconds, and the target substance is an intermediate             filament; and         -   a combination wherein the probe is (d1) a polypeptide,             linked to the luminescent substance, which consists of an             amino acid sequence of sequence number 15, an amino acid             sequence that is a partial amino acid sequence of an amino             acid sequence of sequence number 15, that at least partially             contains an amino acid sequence of 54-557 and that has 556             or fewer amino acids, an amino acid sequence that is a             partial amino acid sequence of an amino acid sequence of             sequence number 15, that at least partially contains an             amino acid sequence of 54-498 and that has 545 or fewer             amino acids, an amino acid sequence that is a partial amino             acid sequence of an amino acid sequence of sequence number             15, that at least partially contains an amino acid sequence             of 167-557 and that has 491 or fewer amino acids, an amino             acid sequence that is a partial amino acid sequence of an             amino acid sequence of sequence number 16, that at least             partially contains an amino acid sequence of 1-251 and that             has 351 or fewer amino acids, an amino acid sequence that is             a partial amino acid sequence of an amino acid sequence of             sequence number 16, that at least partially contains an             amino acid sequence of 3-251 and that has 349 or fewer amino             acids or an amino acid sequence of sequence number 18, (d2)             a polypeptide, linked to the luminescent substance, which             consists of the amino acid sequence described in (d1) where             one or a plurality of amino acids have been substituted,             deleted, inserted or added and for which a half-life of a             probe-target complex formed by binding between the probe and             the target substance is equal to or more than 10             milliseconds and equal to or less than 3 seconds, or (d3) a             polypeptide, linked to the luminescent substance, which             consists of an amino acid sequence having at least a 70%             identity with the amino acid sequence described in (d1) and             for which a half-life of a probe-target complex formed by             binding between the probe and the target substance is equal             to or more than 10 milliseconds and equal to or less than 3             seconds, and the target substance is a focal adhesion.             (7) The method according to (1), wherein     -   the probe contains an antibody or a fragment of an antibody, the         antibody or the fragment being to the target substance and the         antibody or the fragment being linked to the luminescent         substance.         (8) The method according to (7), wherein the fragment of the         antibody is a Fab fragment.         (9) A probe used for labeling a target substance, wherein     -   the probe contains a luminescent substance that emits light         under a prescribed condition,     -   the probe can repeatedly bind to and dissociate from the target         substance directly and specifically, and     -   a half-life of a probe-target complex formed by binding to the         target substance is equal to or more than 10 milliseconds and         equal to or less than 3 seconds.         (10) The probe according to (9), wherein     -   the target substance is an actin polymer and the probe is (a1) a         polypeptide, linked to the luminescent substance, which consists         of an amino acid sequence of sequence number 19, (a2) a         polypeptide, linked to the luminescent substance, which consists         of the amino acid sequence described in (a1) where one or a         plurality of amino acids have been substituted, deleted,         inserted or added and for which a half-life of a probe-target         complex formed by binding between the probe and the target         substance is equal to or more than 10 milliseconds and equal to         or less than 3 seconds, or (a3) a polypeptide, linked to the         luminescent substance, which consists of an amino acid sequence         having at least a 70% identity with the amino acid sequence         described in (a1) and for which a half-life of a probe-target         complex formed by binding between the probe and the target         substance is equal to or more than 10 milliseconds and equal to         or less than 3 seconds,     -   the target substance is a microtubule and the probe is (b1) a         polypeptide, linked to the luminescent substance, which consists         of an amino acid sequence that is a partial amino acid sequence         of an amino acid sequence of sequence number 12, that at least         partially contains an amino acid sequence of 3-309 and that has         407 or fewer amino acids, an amino acid sequence that is a         partial amino acid sequence of an amino acid sequence of         sequence number 14, that at least partially contains an amino         acid sequence of 2536-2843 and that has 408 or fewer amino         acids, an amino acid sequence that is a partial amino acid         sequence of an amino acid sequence of sequence number 14, that         at least partially contains an amino acid sequence of 2781-2819         and that has 138 or fewer amino acids, an amino acid sequence         that is a partial amino acid sequence of an amino acid sequence         of sequence number 4, that at least partially contains an amino         acid sequence of 1-908 and that has 1008 or fewer amino acids,         an amino acid sequence that is a partial amino acid sequence of         an amino acid sequence of sequence number 4, that at least         partially contains an amino acid sequence of 659-908 and that         has 394 or fewer amino acids, an amino acid sequence of sequence         number 5 or an amino acid sequence of sequence number 6, (b2) a         polypeptide, linked to the luminescent substance, which consists         of the amino acid sequence described in (b1) where one or a         plurality of amino acids have been substituted, deleted,         inserted or added and for which a half-life of a probe-target         complex formed by binding between the probe and the target         substance is equal to or more than 10 milliseconds and equal to         or less than 3 seconds, or (b3) a polypeptide, linked to the         luminescent substance, which consists of an amino acid sequence         having at least a 70% identity with the amino acid sequence         described in (b1) and for which a half-life of a probe-target         complex formed by binding between the probe and the target         substance is equal to or more than 10 milliseconds and equal to         or less than 3 seconds,     -   the target substance is an intermediate filament and the probe         is (c1) a polypeptide, linked to the luminescent substance,         which consists of an amino acid sequence that is a partial amino         acid sequence of an amino acid sequence of sequence number 8,         that at least partially contains an amino acid sequence of         3777-4684 and that has 1008 or fewer amino acids, an amino acid         sequence that is a partial amino acid sequence of an amino acid         sequence of sequence number 8, that at least partially contains         an amino acid sequence of 3777-4364 and that has 688 or fewer         amino acids, an amino acid sequence that is a partial amino acid         sequence of an amino acid sequence of sequence number 8, that at         least partially contains an amino acid sequence of 3777-4313 and         that has 637 or fewer amino acids or an amino acid sequence that         is a partial amino acid sequence of an amino acid sequence of         sequence number 8, that at least partially contains an amino         acid sequence of 4022-4364 and that has 443 or fewer amino         acids, (c2) a polypeptide, linked to the luminescent substance,         which consists of the amino acid sequence described in (c1)         where one or a plurality of amino acids have been substituted,         deleted, inserted or added and for which a half-life of a         probe-target complex formed by binding between the probe and the         target substance is equal to or more than 10 milliseconds and         equal to or less than 3 seconds, or (c3) a polypeptide, linked         to the luminescent substance, which consists of an amino acid         sequence having at least a 70% identity with the amino acid         sequence described in (c1) and for which a half-life of a         probe-target complex formed by binding between the probe and the         target substance is equal to or more than 10 milliseconds and         equal to or less than 3 seconds, or     -   the target substance is a focal adhesion and the probe is (d1) a         polypeptide, linked to the luminescent substance, which consists         of an amino acid sequence of sequence number 15, an amino acid         sequence that is a partial amino acid sequence of an amino acid         sequence of sequence number 15, that at least partially contains         an amino acid sequence of 54-557 and that has 556 or fewer amino         acids, an amino acid sequence that is a partial amino acid         sequence of an amino acid sequence of sequence number 15, that         at least partially contains an amino acid sequence of 54-498 and         that has 545 or fewer amino acids, an amino acid sequence that         is a partial amino acid sequence of an amino acid sequence of         sequence number 15, that at least partially contains an amino         acid sequence of 167-557 and that has 491 or fewer amino acids,         an amino acid sequence that is a partial amino acid sequence of         an amino acid sequence of sequence number 16, that at least         partially contains an amino acid sequence of 1-251 and that has         351 or fewer amino acids, an amino acid sequence that is a         partial amino acid sequence of an amino acid sequence of         sequence number 16, that at least partially contains an amino         acid sequence of 3-251 and that has 349 or fewer amino acids, or         an amino acid sequence of sequence number 18, (d2) a         polypeptide, linked to the luminescent substance, which consists         of the amino acid sequence described in (d1) where one or a         plurality of amino acids have been substituted, deleted,         inserted or added and for which a half-life of a probe-target         complex formed by binding between the probe and the target         substance is equal to or more than 10 milliseconds and equal to         or less than 3 seconds or (d3) a polypeptide, linked to the         luminescent substance, which consists of an amino acid sequence         having at least a 70% identity with the amino acid sequence         described in (d1) and for which a half-life of a probe-target         complex formed by binding between the probe and the target         substance is equal to or more than 10 milliseconds and equal to         or less than 3 seconds.         (11) The probe according to (9), wherein     -   the probe contains an antibody or a fragment of an antibody, the         antibody or the fragment being to the target substance and the         antibody or the fragment being linked to the luminescent         substance.         (12) The probe according to (11), wherein     -   the fragment of the antibody is a Fab fragment.         (13) A reagent kit for labeling a target substance, wherein     -   the reagent kit at least includes the probe according to (9).         (14) A screening method of a site in which identifies a target         substance in the probe according to (9), the screening method         comprising:     -   an immobilization step in which a candidate substance of the         site or a substance partially containing the candidate substance         is fixed to a solid support;     -   an observation step in which a target substance linked to a         luminescent substance and a solid support obtained in the         immobilization step are observed in a medium while the target         substance linked to a luminescent substance and the solid         support obtained in the immobilization step are kept in contact,         in a condition that allows observation, in units of 1 molecule,         of light emission from the luminescent substance in a         probe-target complex formed by binding between the target         substance and the candidate substance, and     -   a screening step in which the candidate substance resulting in a         half-life of the probe-target complex that is equal to or more         than 10 milliseconds and equal to or less than 3 seconds is         selected as the site on the basis of observation in the         observation step.         (15) The method according to (14) wherein     -   the candidate substance is an antibody or a fragment of an         antibody from a library of hybridoma that produces an antibody         to the target substance, and     -   the antibody is fixed to a solid support in the immobilization         step.

The present document incorporates the contents of the disclosure of Japanese patent application No. 2015-048692, on the basis of which the priority is claimed for the present application.

According to the present invention, operations and effects that are remarkably more advantageous than those of existing super-resolution microscopy can be provided because (i) the problem of a labeling density, which has been the cause of reduced reliability of existing super-resolution microscopy, can be resolved and (ii) it is easy to visualize a plurality of target substances by protein-based exchangeable probes and there are no limitations on the number of target substances.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be more apparent from the following detailed description when the accompanying drawings are referenced.

FIG. 1 explains the outline of the localization microscopy. FIG. 1 (a) shows the distribution of the actual probe molecules. FIG. 1 (b) shows speckle images containing fluorescent speckles picked up at different times. FIG. 1 (c) shows position information of speckles in the respective speckle images. FIG. 1 (d) shows an observation image reconstructed on the basis of the position information.

FIG. 2 (a) is a schematic view for explaining the principle by which the labeling density can be increased by using the method of the present invention. FIG. 2 (b) is a schematic view for explaining the principle of STORM.

FIG. 3 is a schematic view for explaining procedures for observing a plurality of target substances in one sample by using the method of the present invention.

FIG. 4 is a functional block diagram of a microscope apparatus used for implementing the method of the present invention.

FIG. 5 are related to super-resolution microscopy according to the present invention. Super-resolution microscopy according to the present invention will be referred to as IRIS (image reconstruction by integrating exchangeable single-molecule localization). FIG. 5 (a) shows the outline of IRIS. Transient associations of single-molecule fluorescent probes with their targets are visualized, and central positions of the probes are identified with nanometer accuracy. Integrating the position information from many frames produces a super-resolution image of the target substance. FIG. 5 (b) shows a super-resolution image of a single actin filament in vitro by TIRF (total internal reflection fluorescence) microscopy using Atto 488-Lifeact. An observation image was reconstructed by using high-brightness speckles in order to improve the localization accuracy (see FIG. 9c ). FIG. 5 (c) shows the cross-sectional profile of single actin filaments (n=10 filaments, gray bars). The black curve shows the Gaussian fit to the mean profile, with a FWHM of 23 nm. Error bars show S.E.M. FIG. 5 (d) shows dependency of image quality on the labeling density in vitro. The labeling density of Atto 488-Lifeact per unit length is indicated at the top of each view. FIG. 5 (e) shows line profiles of the labeling density of Atto 488-Lifeact along the frame in FIG. 5 (d). The labeling density is shown after being normalized by the mean labeling density of an entire actin filament. FIG. 5 (f) shows an IRIS image of actin filaments in a cell using Atto 488-Lifeact and total internal reflection illumination. The image was reconstructed from 5×10⁵ frames. FIG. 5 (g) shows a comparison between the IRIS super-resolution image (upper left) and the added SiMS image (lower left) of the region in the frame in FIG. 5 (f). The right graph shows cross-sectional profiles of two adjacent filaments between the two arrowheads in the upper left image. Atto 488-Lifeact was excited by the simultaneous irradiation with 473-nm and 488-nm laser beams in the TIRF mode to obtain a strong signal from each speckle. The stage drift of the microscope was corrected with a bright-field image of nonfluorescent beads in observation of an actin filament in vitro and was corrected with a bright-field image of the cell itself in observation of an actin filament in the cell (see “Methods” in the examples).

FIG. 6 (a) shows super-resolution images of actin filaments using Lifeact. FIG. 6 (b) shows super-resolution images of microtubules using a CLIP-170 fragment (amino acid residues 3-309 of sequence number 12). FIG. 6 (c) shows super-resolution images of intermediate filaments using a Plecin-1 (PLEC) fragment (amino acid residue 4022-4364 of sequence number 8). FIG. 6 (d) shows a super-resolution image of a focal adhesion using a phosphatidylinositol-(4)-phosphate 5-kinase type Iγ-90 (PIPKIγ) fragment (641-668) (sequence number 18). Except for a case when a PIPKIγ fragment was used as a probe, SiMS images (speckle images) of individual probes were obtained by alternately conducting total internal reflection illumination and epi-illumination. Specifically, imaging (exposure time was 50 ms/frame, 20 Hz) for 250 consecutive frames of speckle images based on the total internal reflection fluorescence in accordance with the condition explained in “procedures for imaging of multicolor super-resolution by IRIS” in “methods” of examples and imaging (exposure time was 50 ms/frame, 20 Hz) for 250 consecutive frames of speckle images based on the epi-fluorescence were repeated for observation of actin filament, microtubule and intermediate filament. For imaging of focal adhesions, speckle images based on epi-illumination were not obtained and imaging (exposure time was 50 ms/frame, 20 Hz) of 500 consecutive frames based on total internal reflection illumination was repeated. Then, the super-resolution images in the cell bottom (TIRF=total internal reflection fluorescence) and the entire cell peripheral regions (Epi=epi-fluorescence) were merged so as to reconstruct a super-resolution image. In FIG. 6 (a) through FIG. 6 (c), images obtained by merging TIRF images and epi-fluorescence images are shown, and the enlarged images of the regions enclosed by the frames are shown as the rightmost images. FIG. 6 (e) shows an image resulting from merging seven super-resolution images. The images were respectively reconstructed from 2×10⁵ frames (Lifeact (TIRF), Lifeact (Epi)), 4×10⁴ frames (CLIP frag. (TIRF), CLIP frag. (Epi)), 1.2×10⁵ frames (PLEC frag. (TIRF), PLEC frag. (Epi)) and 4×10⁴ frames (PIPKIγ frag. (TIRF)). The number of frames of speckle images used for constructing the TIRF image of FIG. 6 (a) is 2×10⁵, the number of frames of speckle images used for constructing the Epi image of FIG. 6 (a) is 2×10⁵, the number of frames of speckle images used for constructing the TIRF image of FIG. 6 (b) is 4×10⁴, the number of frames of speckle images used for constructing the Epi of FIG. 6 (b) is 4×10⁴, the number of frames of speckle images used for constructing the TIRF image of FIG. 6 (c) is 1.2×10⁵, the number of frames of speckle images used for constructing the Epi of FIG. 6 (c) is 1.2×10⁵, and the number of frames of speckle images used for constructing the TIRF image of FIG. 6 (d) is 4×10⁴. Also, the probe concentration in the imaging solution for speckle imaging was 2.4 nM for the imaging in FIG. 6 (a), 8 nM for the imaging in FIG. 6 (b), 9.2 nM for the imaging in FIG. 6 (c) and 84 nM for the imaging in FIG. 6 (d). Note that the above probe concentrations are the actual probe concentration in the case when Atto488-Lifeact was used as the probe and are conversion values based on the fluorescence intensity of labeling fluorescent proteins in the case when other probes were used.

FIG. 7 show region-specific proximity between cytoskeletons and focal adhesions. FIG. 7(a) shows an epi-fluorescence super-resolution image of intermediate filaments in the lamella region in ROI1 in FIG. 6 (e). FIG. 7 (b) shows a merged super-resolution image of the intermediate filaments (IF) and actin filaments (Act). FIG. 7 (c) shows a merged super-resolution image of intermediate filaments (IF) and microtubules (MT). FIG. 7 (d) shows a merged super-resolution of intermediate filaments (IF), actin filaments (Act) andmicrotubules (MT) in super-resolution image (a). FIG. 7 (e) shows cross-sectional profiles of three types of cytoskeletons between the arrowheads in FIG. 7 (d). Intermediate filaments are tangled with actin stress fibers (at the positions of arrows) in the lamellar region but are not tangled with microtubules. FIG. 7 (f) shows a TIRF super-resolution image of intermediate filaments in the peripheral region of ROI2 in FIG. 6 (e). FIG. 7 (g) shows a merged TIRF super-resolution image of intermediate filaments (IF) and actin filaments (Act). FIG. 7 (h) shows a merged TIRF super-resolution image of intermediate filaments (IF) and microtubules (MT). FIG. 7 (i) shows a merged TIRF super-resolution image of intermediate filaments (IF), actin filaments (Act), microtubules (MT) and focal adhesions (FA) in the region in the frame in super-resolution image (f). In FIG. 7 (g), which shows a TIRF super-resolution image, the intermediate filaments (IF) are denoted by narrow arrowheads and the actin filaments (Act) are denoted by thick arrowheads. In FIG. 7 (h), which shows a TIRF super-resolution image, the intermediate filaments (IF) are denoted by narrow arrowheads and the microtubules (MT) are denoted by thick arrowheads. FIG. 7 (j) shows cross-sectional profiles of three types of cytoskeletons between the arrowheads in FIG. 7 (i). In this peripheral region, the intermediate filaments overlap a microtubule at the position of the arrow but do not overlap actin filaments.

FIG. 8 show movements of microtubule tips in the vicinity of actin stress fibers and focal adhesions. The left image of FIG. 8 (a) is an image resulting from merging a z position map (FIG. 16) of microtubules (MT) and a TIRF super-resolution image of a focal adhesion (FA) in the central region in FIG. 7 (i). The right image of FIG. 8 (a) is an image resulting from merging a z position map (FIG. 16) of microtubules (MT) and a TIRF super-resolution image of actin filaments (Act) in the central region in FIG. 7 (i). The z positions of microtubules were calculated from the signal intensity ratio between an epi-fluorescence super-resolution image and a TIRF super-resolution image (see “Methods” in the examples). In the left image of FIG. 8 (a), the microtubules (MT) are denoted by arrows and the focal adhesions (FA) are denoted by narrow arrowheads. In the right image of FIG. 8 (a), the microtubules (MT) are denoted by arrows and the actin filaments (Act) are denoted by narrow arrowheads. FIG. 8 (b) shows the line profiles (CLIP frag) of the z position of a microtubule, the intensity (Lifeact) of an actin filament and the intensity (PIPKIγ) of a focal adhesion along the interval between S and E of the microtubules denoted by the thick arrowheads in FIG. 8 (a). The curve of the thick solid line shows a movement average of four data points for the z position of the microtubule. The left image of FIG. 8 (c) is a map of the z positions of microtubules, and the right image of FIG. 8 (c) is an image resulting from merging the TIRF super-resolution images of actin filaments (grey) and focal adhesions (magenta). The focal adhesion was visualized by combining a super-resolution image obtained by using the Src fragment (residues 3-251 of sequence number 15) as a probe and a super-resolution image obtained by using the Paxillin full length as a probe. In FIG. 8 (d), the left panels are live-cell epi-fluorescence observation images (red) and TIRF observation images (green) of EB1-EGFP obtained before IRIS imaging. The middle panels are results of causing overlap between the z position of the tip of the EB1-labeled microtubule, the actin filament and focal adhesion shown in the right image of FIG. 8 (c), and the right panels are results of causing overlap between the speed of the portion of the tip of the EB1-labeled microtubule, the actin filament and focal adhesion shown in the right image of FIG. 8 (c). The microtubule analyzed with the trajectory of EB1 is indicated by an asterisk in FIG. 8 (c).

FIG. 9 show characteristic evaluation of Atto 488-Lifeact. FIG. 9 (a) shows speckle lifetime distribution of Atto488-Lifeact in fixed XTC cells. The SiMS images (speckle images) were picked up consecutively with an exposure time of 10 ms/frame and at a frame rate of 100 Hz by using a 488 nm laser (with the main body output of 50 mW but reaching the sample after being attenuated by AOTF etc.). The measurement of the speckle lifetime (having obtained 100 pixels×100 pixels) of a narrow scope of the sample was performed using the Speckle TrackerJ plug-in so that 100 Hz is achieved, i.e. so that 1 frame was able to be obtained per 10 ms. The black line shows a half-life of 23 ms fit to the single exponential curve of the lifetime distribution of speckles between 20 ms and 110 ms. The photobleaching rate of Atto488-Lifeact was negligible in the term of 200 ms. FIG. 9b shows analysis of Atto488-Lifeact speckles using DAOSTORM (circles in the image). The SiMS images of Atto488-Lifeact in a cell were obtained with an exposure time of 50 ms. The central position of each speckle was determined with nanometer accuracy. The distribution of speckles changed greatly in the frame next to a frame in which an image was picked up 50 milliseconds later. FIG. 9 (c) shows super-resolved images of a single actin filament in vitro by TIRF microscopy using Atto488-Lifeact. The left image is an image obtained by the reconstruction from only high-brightness speckles (top 12% of all measured speckles) and is the same as that shown in FIG. 5 (b). The right image was reconstructed from all speckles. The labeling density on the actin filament is indicated at the top of each image. FIG. 9 (d) shows the cross-sectional profiles of single actin filaments in vitro (n=10 filaments) in the image reconstructed from high-brightness speckles (black) or in the image reconstructed from all speckles (white). Normalization was conducted so that all counts became 1. The black curve shows a Gaussian fit curve with a FWHM of 23 nm (high-brightness speckles) and the white curve shows a Gaussian fit curve with a FWHM of 38 nm (all speckles). The error bar represents ±S.E.M.

FIG. 10 show a comparison of labeling patterns along the longitudinal direction of microtubules between IRIS and other super-resolution techniques. FIG. 10 (a) shows super-resolution images of microtubules by IRIS using CLIP-170 as probes. These images are part of the epi-fluorescence IRIS observation image of FIG. 6 (b). FIG. 10 (b) shows line profiles of labeling intensity along microtubules in the right image of FIG. 10 (a) (assumed to be IRIS (a)), the super-resolution of a microtubule by STORM using the anti-β tubulin described in document 21 (assumed to be STORM (b)), the super-resolution image of a microtubule by STORM using the anti-β tubulin described in document 22 (assumed to be STORM (c)), and the super-resolution image of a microtubule by Exchange-PAINT using the anti-3 tubulin described in document 23 (assumed to be PAINT (d)). The labeling density is shown after being normalized by the mean labeling density of an entire actin filament.

FIG. 11 shows the lifetime distribution data of the selected IRIS probes on the targets. 100 to 112 lifetimes were plotted in accordance with a complementary cumulative distribution function (1-Ndissociation) for each probe. Ndissociation is the cumulative relative frequency of dissociated probes. The half-life of probes on the targets was determined by fitting the life time distribution data by a single exponential function. Part of the measurement results shown on table 1 is shown here.

FIG. 12 show labeling patterns of microtubules by a CLIP-170 fragment (amino acid residues 3-309 of sequence number 12), an APC fragment (amino acid residues 2536-2843 of sequence number 14), a MAP4 fragment (amino acid residues 1-908 of sequence number 4) and Tau isoform 3 (full length of sequence number 5). The patterns are different depending upon procedures for fixing cells. FIG. 12 (a) and FIG. 12 (b) show live-cell imaging during the fixation operation. In FIG. 12 (a), the accumulation of EB1-EGFP at the tips of microtubules disappeared in a process by 3.7% PFA and 0.5% Triton-X 100. In FIG. 12 (b), EB1-EGFP was fixed in a state in which it was accumulated at the tips of the microtubules in a process by cold methanol. FIG. 12 (c) shows TIRF IRIS images (observation images) of microtubules using CLIP-170 fragment and Tau isoform 3 in cells processed by PFA and Triton-X 100. With these probes, IRIS images of the entire microtubules were obtained. The image resulting from merging these IRIS images indicates that the entire microtubules visualized by these probes correspond to each other. FIG. 12 (d) shows the TIRF IRIS images of microtubules using a CLIP-170 fragment and an APC fragment in cells preprocessed with cold methanol for 5 seconds and next with PFA and Triton X-100 as a preprocess. These probes resulted in IRIS images of microtubules where the microtubule tips were visualized with much stronger signals than were the entire microtubules. The image resulting from merging these images shows a coincidence of the microtubule tips visualized by these probes. FIG. 12 (e) shows TIRF IRIS images of microtubules using a CLIP-170 fragment, a MAP4 fragment and Tau isoform 3 in cold methanol-preprocessed cells. The microtubule tips were labeled strongly with a CLIP-170 fragment while they were weakly labeled with a MAP4 fragment and Tau isoform 3 (see merged images in the lower tier).

FIG. 13 show labeling patterns of focal adhesions by Paxillin full length (FL) (full length of sequence number 15), the Src fragment (amino acid residues 3-251 of sequence number 16) and a PIPKIγ fragment (residues 641-668) (sequence number 18). FIG. 13 (a) shows live-cell images in which GFP-Paxillin was used for visualizing focal adhesions for comparison. FIG. 13 (b) shows images of cells obtained by fixing and providing a permeabilization process to the living cells after the imaging of FIG. 13 (a). FIG. 13 (c) shows a state in which a remaining fluorescence of GFP-Paxillin was completely bleached by irradiation with a strong excitation laser. FIG. 13 (d) through FIG. 13 (f) are IRIS images in the order of the Src fragment, Paxillin FL, and a PIPKIγ fragment after the photobleaching of FIG. 13 (c). In an IRIS image view of FIG. 13 (d), generated by using the Src fragment, some dot structures appeared (arrowheads in FIG. 13 (d)) in the focal adhesion. These dot structures were not reconstructed by probes that did not dissolve, but were reconstructed by probes that repeatedly bound. In Paxillin FL, a focal adhesion was labeled partially. The left image of FIG. 13 (g) is a result of merging FIGS. 13 (a) (d) (e) and (f), the center image is a result of merging FIGS. 13 (d) and (e), and the right image is a result of merging FIGS. 13 (d) and (f). In the center image, the dot structures visualized by Src fragments (portions denoted by the arrowheads) have many portions that do not correspond to the focal adhesions visualized by Paxillin FL. FIG. 13 (h) shows the cross-sectional profiles of the focal adhesions in three IRIS images and the living-cell image of GFP-Paxillin. The measured scope is denoted by the line in the left image of FIG. 13 (g). The dot structures visualized by Src fragments are not completely visualized by Paxillin FL (arrow in FIG. 13 (h)). These proteins have different binding partners in focal adhesions (documents 17, 38 and 39). These differences between IRIS images may indicate a distribution of partners that respective proteins can associate with in focal adhesions.

FIG. 14 show the z profile of the TIRF excitation intensity. FIG. 14 (a) shows fluorescence images of HyLight488-labeled microtubules obtained by total internal reflection fluorescence (TIRF) and epi fluorescence. The epi fluorescence images were obtained as z-stack images (0.2 μm step size). The z-stack epi-fluorescence images were used to determine the z-directional distance of each point along the tilted microtubule (see “Methods”). The bar represents 5 μm. FIG. 14 (b) shows the Z profile of the TIRF excitation intensity.

FIG. 15 (a) shows z-position mapping of actin bundles, FIG. 15 (b) shows z-position mapping of microtubules, and FIG. 15 (c) shows z-position mapping of intermediate filaments. The respective images of FIGS. 15 (a), (b) and (c) were calculated and generated from the total internal reflection fluorescence (TIRF) IRIS image and the epi-fluorescence IRIS image shown in FIGS. 6 (a), (b) and (c) (see “Methods”). The right image is an enlarged image of the region in the frame shown in the left image. FIG. 15 (d) shows a distribution of z positions of actin bundles in lamellipodia (LP), stress fibers (SF) and actin arcs (Arc). In these layered actin structures, a calculated z position represents the height position of the center of gravity in the z axial direction of an actin filament. FIG. 15 (e) shows the z-position profile along the longitudinal direction of a microtubule. The microtubule submerges toward the periphery of a cell (arrowheads in FIG. 15 (b)). S and E in FIG. 15 (b) represent the starting and ending points of the line profile.

FIG. 16 shows an epi-fluorescence super-resolution image of microtubules (left), a TIRF image (center) and the z-position mapping shown in FIG. 8 (a) (right). In CLIP-170 fragment-visualized microtubules, signals were partially lost (asterisks in the left and center images).

FIG. 17 shows a result of IRIS super-resolution imaging using a probe containing a Fab fragment (Fab probe). It is a result of imaging a distribution of p40, which is a subunit of an Arp2/3 complex, by using a Fab probe that is produced from anti-p40 polyclonal antibody.

FIG. 18 show binding of FLAG-EGFP to a solid-phased antibody. An antibody contained in hybridoma culture supernatant was fixed to a glass surface and a FLAG-EGFP solution was added so as to observe it with a TIRF microscope. Each speckle is FLAG-EGFP that has bound to a solid-phased antibody. FIG. 18 (A) shows a reaction positive example. Speckles representing binding between FLAG-EGFP and antibodies are observed at a high density. An observation result of a great number of speckles indicates that solid-phased antibodies are anti-FLAG antibodies having a binding capacity to FLAG-EGFP. FIG. 18 (B) shows a reaction negative example. Few speckles are observed. This is a similar level to a case where FLAG-EGFP was added to a glass surface that was not solid phased.

FIG. 19 shows super-resolution images of FLAG fused actins obtained by using a Fab probe derived from an anti-FLAG monoclonal antibody selected from a hybridoma library (203 milliseconds as the half-life of a probe-target complex). This cell has forcibly expressed a FLAG tag fused actin. The right image is an enlarged image of the region in the white frame in the left image.

DESCRIPTION OF THE EMBODIMENTS

In super-resolution microscopy, an increased resolution capability has brought the new problems of a low labeling density and unevenness thereof. According to a sampling theorem for example, it has been shown that one labeling substance has to be included at 10 nm in a target in order to obtain a spatial resolution capability of 20 nm (J. Cell Sci., 126, 3505-3513, 2013). In conventional super-resolution imaging of proteins in cells, target proteins are labeled by using the expression of target proteins with which fluorescent proteins have been made to fuse or by using a fluorescent antibody. This imposes limitations on a labeling density, depending upon the expression amount ratio to endogenous target proteins and upon the size (about 10 nm) of an antibody itself. Also, because fluorescent dyes that have bound to a target substance are used, the maximum number of types of target substances that can be visualized in one sample is two or three. The limitations on the number of proteins that can be stained have been a long-standing issue in the study of a cell consisting of various types of proteins.

In Exchange-PAINT above as well, target substances have to be labeled with antibodies that were made to fuse with DNA oligomers, and thus it is expected that a plurality of antibodies will interfere with each other spatially in a region at or below the diffraction limit. In principle, the greater the number of types of antibodies there are, the more difficult it is to perform labeling evenly.

Uneven labeling leads to reconstruction of false super-resolution, which is problematic.

Proceedings of the national Academy of Sciences of the United States of America 103, 18911-18916 (2006), which discloses PAINT, does not at all describe increasing of the labeling density of target substances by labeling substances.

Thus, it is an object of the present invention to provide a super-resolution microscope observation method that can obtain position information of luminescent substances at a high density, the luminescent substances being used for labeling.

According to the method of the present invention, it is possible to label target substances at a high density in a sample by using luminescent probes and to generate a highly accurate observation image of a target substance.

1. Principle of IRIS

The present inventors have named the observation method of the present invention IRIS (image reconstruction by integrating exchangeable single-molecule localization).

FIG. 2 (a) explains functions of a luminescent probe in the observation method of the present invention. A probe 200 contains a luminescent substance 201 that emits light under a prescribed condition (for example a fluorescent substance that emits fluorescence when irradiated with excitation light), and usually further contains a binding substance 202 that is involved in binding to and dissociation from a target substance that is linked to the luminescent substance 201. The probe 200 can repeatedly bind to and dissociate from a target substance 210 directly and specifically. Characteristics of binding of the probe 200 are as below. When the probe 200 is in a state in which it has bound to the target substance 210, the light emitted by the luminescent substance 201 can be picked up as a speckle in a speckle image, and when the probe 200 has dissociated from the target substance 210, the light emitted by the luminescent substance 201 is not picked up as a speckle in an image because the probe is in a disordered thermal motion in a medium 220. In an imaging step of the present invention, a step is performed a plurality of times (e.g. hundreds of times through hundreds of thousands of times respectively at different times) in which a speckle image including, as a speckle, light emitted from the luminescent substance 201 under a prescribed condition is obtained in a state in which the medium 220 containing the probe 200 is brought into contact with a sample containing the target substance 210. As is shown when superposing the obtained plurality speckle images, an imaging step of the present invention can attain the same effect as that attained by labeling the target substances 210 with probes 200 at a high density and picking up images of them. Theoretically, there is no upper limit on the density of the labeling in the above. Also, when the concentration of the probes 200 in the medium 220 is adjusted appropriately, it is possible to identify a plurality of individual speckles in a separate manner in one speckle image.

Meanwhile, in PALM, STORM and Exchange-PAINT, which have conventionally been known as localization microscopy, an antibody to which a luminescent substance is linked is used for labeling a target substance. In an example of STORM for example, as shown in FIG. 2 (b), luminescent antibodies 230 resulting from linking luminescent substances 232 and antibodies 231 for the target substance 210 are made to bind to the target substance 210 and images are picked up a plurality of times (e.g. hundreds of times through hundreds of thousands of times) at different times while making the luminescent substances 232 emit light discretely at a density that is low enough to prevent the speckles from overlapping. By superposing the obtained plurality of speckle images, an image with all the luminescent substances 232 emitting light can be obtained. However, the labeling density at which the target substance is labeled with the luminescent antibodies 230 is limited by the size (about 10 nm) of the antibodies themselves. In order to achieve a resolution capability of Xnm, it is necessary to label a target substance at intervals of X/2 nm (Nyquist Sampling Theorem). The luminescent antibody 230 has a size of at least about 10 nm, so making labeling at a high density is impossible. Also, in Exchange-PAINT, a plurality of target substances in one sample are labeled with a plurality of antibodies that are specific to the respective target substances, which causes interference between the plurality of antibodies, making it further difficult to perform labeling at a high density. The observation method according to the present invention can solve the above problem of labeling density in conventional localization microscopy.

2. Observation of a Plurality of Target Substances

The observation method according to the present invention can preferably be implemented even when a plurality of target substances exist in one sample.

When a plurality of target substances exist in one sample, the observation method according to the present invention are implemented through the following procedures. A medium containing a probe that repeatedly binds to and dissociates from one type from among a plurality of target substances directly and specifically is brought into contact with a sample so that an imaging step of the present invention is conducted and thereby a plurality of speckle images including speckles of light emitted from the probes that have bound to that one type of the target substance are obtained. Next, probes are removed by washing the sample and a medium containing a probe that repeatedly binds to and dissociates from one different type from among a plurality of target substances directly and specifically is brought into contact with the sample so that an imaging step of the present invention is conducted and thereby a plurality of speckle images including speckles of light emitted from the probes that have bound to that one different type of the target substance are obtained. These procedures are performed until a plurality of speckle images are obtained for each of all the target substances that are to be observed. The wash of the sample between the respective imaging steps can be implemented by for example performing at least one time an operation in which an appropriate washing medium such as the above medium etc. not containing the probe is brought into contact with the sample the sample is washed. Observation images of respective target substances can be generated from a plurality of speckle images for respective target substances obtained by using respective probes. Also, by superposing observation images of respective target substances in the same sample, a multiple-observation image including observation images of a plurality of target substances in the sample can also be generated.

A probe used in the present invention is a probe that can repeatedly bind to and dissociate from a target substance directly and specifically. “Bind to a target substance directly” used herein refers to binding between a probe and a target substance that is not through another binding substance such as an antibody etc. When a probe and a target substance are binding through at least one type of binding selected from a group consisting of for example hydrogen binding, hydrophilic and/or hydrophobic binding, electrostatic binding and van der Waals binding, the probe and the target substance can be treated as binding directly.

In the present invention, because a probe and a target substance bind to and dissociate from each other directly, the original state of the sample can be recovered when the sample is washed so as to remove the probe after performing an imaging step in which the sample is processed with the probe so as to make the sample emit light. Even in a case when an operation is repeatedly performed sequentially for each target substance, in which a medium containing a probe is brought into contact with a sample, an imaging step is performed and washing is performed in order to obtain observation images of a plurality of target substances included in one sample, and each imaging step can be performed in a state where only a probe for one type of a target substance that is to be observed exists and probes for other target substances do not exist. This prevents interference from occurring between probes and makes it possible to observe each target substance in a sample that is in a more natural state.

FIG. 3 schematically shows procedures for obtaining each observation image by using one sample containing target substances A and target substances B. First, an imaging step is performed in a state where a medium containing probes A that are specific to target substances A is brought into contact with a sample so as to obtain a plurality of speckle images. Next, the sample is washed so as to remove the probes A. Thereafter, an imaging step is performed in a state where a medium containing probes B that are specific to target substances B is brought into contact with the sample so as to obtain a plurality of speckle images. An observation image is generated for each of target substance A and target substance B from a plurality of speckle images for the respective target substances. It is also possible to generate a multiple-observation image by superposing an observation image of target substance A and an observation image of target substance B.

3. Explanations for Sample and Probe

A sample used for observation is typically a biological sample such as a cell, a tissue etc. consisting of a plurality of cells. When a sample is a biological sample, it is preferable to use a sample in a fixed state, and it is particularly preferable to use a sample that has further received a permeabilization process as necessary.

A sample contains at least one target substance. “Target substance” used herein refers to an object, as an observation target, that is contained in a sample. Examples of a target substance may include a structure body that constitutes a cytoskeleton preferably such as an actin polymer, a microtubule, an intermediate filament, a focal adhesion, etc. A target substance is preferably a structure that is, like the above structure, formed as a result of many constituents assembling, the constituents having the same structure or having a structure having common characteristics. Origin organisms for a structure body that constitutes a biological sample such as a cell, a tissue, etc. that can be an observation sample and a cytoskeleton that can be a target substance are not particularly limited. For example, a structure originating from vertebrates such as mammals (humans, rabbits, rodents, etc.), amphibians (frogs etc. xenopus for example), fish including bony fish and cartilaginous fish, reptiles, birds, etc., invertebrates such as mollusks, protochordates, echinoderm, cnidarians, arthropods, etc., and unicellular organisms such as eukaryotic unicellular organisms (yeast etc.) can be used. Among them, structure bodies constituting cytoskeletons are held by a wide variety of species, and a method of observing a structure body constituting a cytoskeleton whose effectiveness has been confirmed in the examples can be applied regardless of the type of origin organism. It is preferable that a target substance be an object having a shape that does not substantially change during an imaging step of picking up a plurality of speckle images, and a target substance may receive a fixation process as necessary.

A target substance is preferably a target substance containing a protein, and is more preferably a protein target substance that exits together with one or more different proteins, which are not the target substance, and that are in an observation target sample. In the present embodiment of the present invention, a probe specifically binds to a protein that is a target substance from among a plurality of types of proteins, making it possible to selectively visualize a target substance.

A probe has a property of repeatedly binding to and dissociating from one specific type of target substances in a sample directly and specifically. The meaning of “binding to a target substance directly” is as mentioned previously.

While a probe may be a luminescent substance that by itself has a property of binding to and dissociating from a target substance, it usually contains a luminescent substance and a binding substance that is linked to that luminescent substance and that has a property of repeatedly binding to and dissociating from a target substance directly and specifically.

A probe and a target substance have the following binding characteristics. Specifically, the half-life of a probe-target complex formed by binding between a probe and a target substance is preferably equal to or more than 10 milliseconds and equal to or less than 3 seconds, more preferably equal to or more than 10 milliseconds and equal to or less than 2 seconds, more preferably equal to or more than 10 milliseconds and equal to or less than 1 second, more preferably equal to or more than 10 milliseconds and equal to or less than 900 milliseconds, more preferably equal to or more than 10 milliseconds and equal to or less than 800 milliseconds, more preferably equal to or more than 10 milliseconds and equal to or less than 700 milliseconds, more preferably equal to or more than 10 milliseconds and equal to or less than 600 milliseconds, more preferably equal to or more than 10 milliseconds and equal to or less than 500 milliseconds, more preferably equal to or more than 20 milliseconds, and more preferably equal to or less than 300 milliseconds, particularly preferably equal to or less than 250 milliseconds. The above half-life herein is preferably a half-life of a probe-target complex formed by binding between a probe and a target substance in a case when a medium containing a probe and a sample containing a target substance that is to be observed are brought into contact under a condition for performing an imaging step. The half-life is defined by a period of time before the number of probes that have bound to target substances at a given moment is reduced to half through dissociation. Measurement procedures for the half-life are as below. A medium containing a probe is brought into contact with a sample containing a target substance that is to be observed and a period of time between when light emission speckle based on the probe appears and when it disappears is measured for each light emission speckle while performing observation under a condition that is used for observation. Then, periods of time between the appearance and disappearance of the speckles are plotted in accordance with a complementary cumulative relative frequency function (1-Ndissociation). Ndissociation is a cumulative relative frequency of a probe that dissociated. A cumulative relative frequency is a ratio of the speckles that disappeared within a given period of time to the total number of measured speckles (i.e. probes) (i.e. probes that dissociated within a given period of time), and represents the total number as 1. Then, by fitting the above complementary cumulative relative frequency function with an exponent function, that half-life is calculated.

The period of time between the appearance and disappearance of a light emission speckle based on a probe can be measured by the following procedures. Specifically, while keeping a medium containing a probe in contact with a target substance and providing a prescribed condition necessary for emitting light (for example irradiation with excitation light), speckle images including speckles based on the light emission of luminescent substances of probes are consecutively picked up with an exposure time of X seconds (for example 0.050 seconds, 0.100 seconds, etc.) and at a frame rate of 1/XHz. Then, the period of time is measured between when a probe that has bound to a target appears in a speckle image and when it disappears through dissociation. In the above, when observation is performed for Y consecutive frames, and for a speckle not observed in frames before and after them, the period of time between the appearance and disappearance of that speckle can be treated as YX seconds.

A probe with the above half-life equal to or more than 10 milliseconds provides a period of time between binding to and dissociation from a target substance that is sufficiently long to allow an ordinary highly sensitive imaging device such as an EM-CCD camera etc. to pick up an image of a speckle based on the luminescent substance of the probe that has been bound. When by contrast the half-life is too long, a region including a probe having a long binding time to a target substance represents a signal that is particularly strong in a reconstructed observation image, which can cause uneven labeling. This makes it difficult to obtain an accurate distribution of target substances. From the results obtained thus far, it is possible to obtain a speckle image and an observation image that are relatively even when a probe whose half-life is equal to or less than 3 seconds is used and the shorter the half-life of a probe that is used is, the easier it is to obtain a speckle image and an observation image that are even. This point of view has made obvious that a half-life that is equal to or less than 500 milliseconds is particularly preferable. Also, as a general rule, the longer a half-life is, the longer an exposure time for one frame and imaging intervals have to be, which sometimes requires a long period of time for obtaining speckle images that are needed for generating a reconstruction image. From this point of view, it is preferable that the half-life be equal to or less than 3 seconds or that the half-life be further shorter.

In the present invention, a step of obtaining each speckle image will be referred to as a “frame imaging step”. In each frame imaging step, a speckle image including a speckle of light emitted from a luminescent substance under a prescribed condition is picked up by using an imaging device. A term in which one speckle image is picked up will be referred to as a “frame”, a term between the starting time of a frame and the starting time of the next frame will be referred to as an “interval”, and a term between the ending time of a frame and the starting time of the next frame will be referred to as an “inter-frame term”. A period of time for one frame (exposure time) can be determined in accordance with the binding half-life between a probe and a target substance in an appropriate manner. It is preferable that a period of time for one frame (exposure time) be longer than the binding half-life between a probe and a target substance, but the scope of the present embodiment is not limited to this.

Target substances and probes that meet the above requirements can be selected appropriately while a combination between a target substance and a binding substance of a probe is preferably selected from a group of:

-   -   a combination wherein the probe is (a1) a polypeptide, linked to         the luminescent substance, which consists of an amino acid         sequence of sequence number 19, (a2) a polypeptide, linked to         the luminescent substance, which consists of the amino acid         sequence described in (a1) where one or a plurality of amino         acids have been substituted, deleted, inserted or added, and for         which a half-life of a probe-target complex formed by binding         between the probe and the target substance is equal to or more         than 10 milliseconds and equal to or less than 3 seconds, or         (a3) a polypeptide, linked to the luminescent substance, which         consists of an amino acid sequence having at least a 70%         identity with the amino acid sequence described in (a1) and for         which a half-life of a probe-target complex formed by binding         between the probe and the target substance is equal to or more         than 10 milliseconds and equal to or less than 3 seconds, and         the target substance is an actin polymer;     -   a combination wherein the probe is (b1) a polypeptide, linked to         the luminescent substance, which consists of an amino acid         sequence which is a partial amino acid sequence of an amino acid         sequence of sequence number 12, which at least partially         contains an amino acid sequence of 3-309 and which has 407 or         fewer, preferably 357 or fewer, more preferably 327 or fewer and         most preferably 307 amino acids, an amino acid sequence which is         a partial amino acid sequence of an amino acid sequence of         sequence number 14, which at least partially contains an amino         acid sequence of 2536-2843 and which has 408 or fewer,         preferably 358 or fewer, more preferably 328 or fewer and most         preferably 308 amino acids, an amino acid sequence which is a         partial amino acid sequence of an amino acid sequence of         sequence number 14, which at least partially contains an amino         acid sequence of 2781-2819 and which has 138 or fewer,         preferably 88 or fewer, more preferably 58 or fewer and most         preferably 38 amino acids, an amino acid sequence which is a         partial amino acid sequence of an amino acid sequence of         sequence number 4, which at least partially contains an amino         acid sequence of 1-908 and which has 1008 or fewer, preferably         958 or fewer, more preferably 928 or fewer and most preferably         908 amino acids, an amino acid sequence which is a partial amino         acid sequence of an amino acid sequence of sequence number 4,         which at least partially contains an amino acid sequence of         659-908 and which has 394 or fewer, preferably 344 or fewer,         more preferably 314 or fewer and most preferably 294 amino         acids, an amino acid sequence of sequence number 5 or an amino         acid sequence of sequence number 6, (b2) a polypeptide, linked         to the luminescent substance, which consists of the amino acid         sequence described in (b1) where one or a plurality of amino         acids have been substituted, deleted, inserted or added and for         which a half-life of a probe-target complex formed by binding         between the probe and the target substance is equal to or more         than 10 milliseconds and equal to or less than 3 seconds or (b3)         a polypeptide, linked to the luminescent substance, which         consists of an amino acid sequence having at least a 70%         identity with the amino acid sequence described in (b1) and for         which a half-life of a probe-target complex formed by binding         between the probe and the target substance is equal to or more         than 10 milliseconds and equal to or less than 3 seconds, and         the target substance is a microtubule;     -   a combination wherein the probe is (c1) a polypeptide, linked to         the luminescent substance, which consists of an amino acid         sequence which is a partial amino acid sequence of an amino acid         sequence of sequence number 8, which at least partially contains         an amino acid sequence of 3777-4684 and which has 1008 or fewer,         preferably 958 or fewer, more preferably 928 or fewer and most         preferably 908 amino acids, an amino acid sequence which is a         partial amino acid sequence of an amino acid sequence of         sequence number 8, which at least partially contains an amino         acid sequence of 3777-4364 and which has 688 or fewer,         preferably 638 or fewer, more preferably 608 or fewer and most         preferably 588 amino acids, an amino acid sequence which is a         partial amino acid sequence of an amino acid sequence of         sequence number 8, which at least partially contains an amino         acid sequence of 3777-4313 and which has 637 or fewer,         preferably 587 or fewer, more preferably 557 or fewer and most         preferably 537 amino acids, or an amino acid sequence which is a         partial amino acid sequence of an amino acid sequence of         sequence number 8, which at least partially contains an amino         acid sequence of 4022-4364 and which has 443 or fewer,         preferably 393 or fewer, more preferably 363 or fewer and most         preferably 343 amino acids, (c2) a polypeptide, linked to the         luminescent substance, which consists of the amino acid sequence         described in (c1) where one or a plurality of amino acids have         been substituted, deleted, inserted or added and for which a         half-life of a probe-target complex formed by binding between         the probe and the target substance is equal to or more than 10         milliseconds and equal to or less than 3 seconds, or (c3) a         polypeptide, linked to the luminescent substance, which consists         of an amino acid sequence having at least a 70% identity with         the amino acid sequence described in (c1) and for which a         half-life of a probe-target complex formed by binding between         the probe and the target substance is equal to or more than 10         milliseconds and equal to or less than 3 seconds, and the target         substance is an intermediate filament; and (d1) a polypeptide,         linked to the luminescent substance, which consists of an amino         acid sequence of sequence number 15, an amino acid sequence         which is a partial amino acid sequence of an amino acid sequence         of sequence number 15, which at least partially contains an         amino acid sequence of 54-557 and which has 556 or fewer, more         preferably 524 or fewer and most preferably 504 amino acids, an         amino acid sequence which is a partial amino acid sequence of an         amino acid sequence of sequence number 15, which at least         partially contains an amino acid sequence of 54-498 and which         has 545 or fewer, preferably 495 or fewer, more preferably 465         or fewer and most preferably 445 amino acids, an amino acid         sequence which is a partial amino acid sequence of an amino acid         sequence of sequence number 15, which at least partially         contains an amino acid sequence of 167-557 and which has 491 or         fewer, preferably 441 or fewer, more preferably 411 or fewer and         most preferably 391 amino acids, an amino acid sequence which is         a partial amino acid sequence of an amino acid sequence of         sequence number 16, which at least partially contains an amino         acid sequence of 1-251 and which has 351 or fewer, preferably         301 or fewer, more preferably 271 or fewer and most preferably         251 amino acids, an amino acid sequence which is a partial amino         acid sequence of an amino acid sequence of sequence number 16,         which at least partially contains an amino acid sequence of         3-251 and which has 349 or fewer, preferably 299 or fewer, more         preferably 269 or fewer and most preferably 249 amino acids, or         an amino acid sequence of sequence number 18, (d2) a         polypeptide, linked to the luminescent substance, which consists         of the amino acid sequence described in (d1) where one or a         plurality of amino acids have been substituted, deleted,         inserted or added and for which a half-life of a probe-target         complex formed by binding between the probe and the target         substance is equal to or more than 10 milliseconds and equal to         or less than 3 seconds or (d3) a polypeptide, linked to the         luminescent substance, which consists of an amino acid sequence         having at least a 70% identity with the amino acid sequence         described in (d1) and for which a half-life of a probe-target         complex formed by binding between the probe and the target         substance is equal to or more than 10 milliseconds and equal to         or less than 3 seconds, and the target substance is a focal         adhesion. It is also preferable that a more preferable scope of         half-lives of probe-target complexes in the above respective         combinations be in accordance with the above and particularly be         a period of time that is equal to or more than 10 milliseconds         and equal to or less than 500 milliseconds or shorter. In the         above combinations, more preferable scopes for a half-life of a         probe-target complex are as described above and it is         particularly preferable that a half-life of a probe-target         complex be equal to or more than 10 milliseconds and equal to or         less than 500 milliseconds or that a half-life of a probe-target         complex be further shorter.

Origin organisms for an actin polymer, a microtubule, an intermediate filament and/or focal adhesion in the above combinations are not particularly limited, however a substance originating from vertebrates such as mammals (humans, rabbits, rodents, etc.), amphibians (frogs etc. xenopus for example), fish including bony fishes and cartilaginous fishes, reptiles, birds, etc., invertebrates such as mollusks, protochordates, echinoderm, cnidarians, arthropods, etc., a unicellular organism such as eukaryotic unicellular organisms (yeast etc.), and a substance resulting from artificially introducing variation to them can be used.

“Actin polymer” used herein refers to a structure body formed through polymerization of an actin molecule, and typically to an actin filament.

In this document, a “microtubule” refers to for example a structure in which 13 protofilaments where heterodimers of atubulin and (tubulin are connected in a fibrous manner are collected to form a tubular structure body having a diameter of 25 nm.

In this document, intermediate filaments of type I, type II, type III and type IV are known as an “intermediate filament”, and all of them may be observation targets of the present invention. Plectin can bind to all types of intermediate filaments.

In this document, “focal adhesion” refers to a structure made by for example a plurality of proteins (integrin, paxillin, vinculin, talin, etc.) being provided at adhesion points between a cell and an extracellular matrix.

“One or a plurality” for substitution, deletion, insertion or addition of an amino acid in the above (a2), (b2), (c2) and (d2) refers to for example 1 to 50, preferably 1 to 25, more preferably 1 to 20, more preferably 1 to 15, more preferably 1 to 10, more preferably 1 to 7, more preferably 1 to 5, more preferably 1 to 4, more preferably 1 to 3, and most preferably 1 or 2. Further preferably, the above expression of “one or a plurality” refers to preferably equal to or less than 20%, more preferably equal to or less than 10% and most preferably equal to or less than 5% of the number of amino acids of the polypeptides in the above (a1), (b1), (c1) and (d1).

Substitution of an amino acid is preferably conservative amino acid substitution. “conservative amino acid substitution” refers to substitution between amino acids having similar physicochemical functions such as an electric charge, a side chain, a polarity, an aromatic property, etc. Amino acids having similar physicochemical functions can be categorized into for example a basic amino acid (arginine, lysine, histidine), an acidic amino acid (aspartic acid, glutamic acid), a non-charged polar amino acid (asparagine, glutamine, serine, threonine, cysteine, tyrosine), a non-polar amino acid (glycine, leucine, isoleucine, alanine, valine, proline, phenylalanine, tryptophan, methionine), a branched-chain amino acid (leucine, valine, isoleucine), an aromatic amino acid (phenylalanine, tyrosine, tryptophan, histidine), etc. Addition of one or a plurality of amino acids in each amino acid sequence is preferably addition of a total of one or a plurality of amino acids to at least one of the N-terminus and the C-terminus of that amino acid sequence. Also, deletion of one or a plurality of amino acids in each amino acid sequence is preferably deletion of a total of one or a plurality of amino acids from at least one of the N-terminus and the C-terminus of that amino acid sequence.

Identity with the amino acid sequences respectively described in the above (a1), (b1), (c1) and (d1) in the above (a3), (b3), (c3), and (d3) is preferably equal to or more than 80%, more preferably equal to or more than 85%, more preferably equal to or more than 90%, more preferably equal to or more than 95%, more preferably equal to or more than 97%, more preferably equal to or more than 98%, and most preferably equal to or more than 99%. In the present invention, the value of identity of amino acid sequences is calculated in a default setting by using software that computes identity between a plurality of amino acid sequences (for example FASTA, DANASYS and BLAST). The value of identity of amino acid sequences is calculated by calculating the number of amino acid residues that match when a pair of amino acid sequences is aligned in such a manner that the matching degree becomes the maximum and is calculated as a ratio of the number of the matching amino acid residues to the total number of the amino acid residues of the amino acid sequences that were compared. In this example, when there are gaps, the above total number of the amino acid residues is the number of amino acid residues obtained by counting one gap as one amino acid residue. When thus calculated, the total numbers of all the amino acid residues are different between the amino acid sequences that are compared, and the identity is calculated on the basis of the greater total number of the amino acid residues. For a detailed method of determining identity, reference is to be made to for example Altschul et al, Nuc. Acids. Res. 25, 3389-3402, 1977 and Altschul et al, J. Mol. Biol. 215, 403-410, 1990.

Also, more preferably, polypeptides in the above (a3), (b3), (c3) and (d3) are polypeptides having preferably equal to or more than 75%, more preferably equal to or more than 80%, more preferably equal to or more than 85%, more preferably equal to or more than 90%, more preferably equal to or more than 95%, more preferably equal to or more than 97%, more preferably equal to or more than 98%, and most preferably equal to or more than 99%, of similarly to the amino acid sequences respectively described in the above (a1), (b1), (c1) and (d1). The value of similarity of amino acid sequences is calculated by calculating the total of the number of amino acid residues that match when a pair of amino acid sequences is aligned in such a manner that the matching degree becomes the maximum and the amino acid residues have similar physicochemical functions, and is calculated as a ratio of the total number to the total number of the amino acid residues of the amino acid sequences that were compared. In this example, similarity of amino acid sequences can be calculated by a computer by using software similar to that described for the identity of amino acid sequences. A method of calculating the total number of amino acid residues is as described above for amino acid identity. The meaning of amino acid residues having similar physicochemical functions is as described above.

In a preferable embodiment of the present invention, a binding substance contained in a probe (a site for identification of a target substance) is an antibody or a fragment of an antibody, to a target substance, and particularly preferably a fragment of an antibody.

A technique for producing an antibody to an arbitrary target substance has already been established. Thus, using an antibody or a fragment of an antibody produced in accordance with a target substance as a binding substance of a probe makes it possible to use the technique of the present invention for observation of a target substance ranging in a wide scope.

It is sufficient if a target substance in this embodiment is a target substance presenting antigenicity, and it is typically a protein.

An antibody that an antibody or a fragment of an antibody contained in a probe is from is typically immunoglobulin G (IgG), however a different isotype is possible, and it may be for example immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulinA (IgA), immunoglobulin E (IgE), etc. When these types of immunoglobulin contain a plurality of subclasses, one belonging to an arbitrary subclass is possible. Examples of another type of antibody may include a single domain antibody. These antibodies may be a variation or may be in a form in which it is made to fuse with a different polypeptide.

Although origins of an antibody are not particularly limited, for example, an antibody originating from for example nonhuman animals such as mice, rats, llamas, camels, etc. and from humans etc. can be used. Also, an antibody may be a chimeric antibody formed by causing a fusion of domains of antibodies of a plurality of origins.

An antibody may be a polyclonal antibody or may be a monoclonal antibody.

“Fragment” of an antibody in the present embodiment refers to a “functional fragment” having avidity to a target substance (antigen). A fragment of an antibody may be a variant or may be in a form in which it is made to fuse with a different polypeptide.

Examples of a fragment of an antibody may include a Fab fragment, a Fab′ fragment, a F (ab′)₂ fragment, an scFv (single-strand Fv) fragment, a VHH fragment of a single domain antibody (for example a commercial name of a nanobody), etc. A Fab fragment can be obtained by cutting an antibody with papain, a protein breakdown enzyme. A F (ab′)₂ fragment can be obtained by cutting an antibody with pepsin. A Fab′ fragment can be obtained by further processing a F (ab′)₂ fragment under a reduction condition. An scFv fragment is a result of linking a heavy-chain variable region and a light-chain variable region of an antibody with a linker of a polypeptide so as to make them single-stranded and can be produced by a gene engineering method that utilizes a polynucleotide having a base sequence that encodes the heavy-chain variable region, the linker and a light-chain variable region.

In the present embodiment, the half-life of a probe-target complex formed by binding of a probe containing an antibody or a fragment of an antibody to a target substance is equal to or more than 10 milliseconds and equal to or less than 3 seconds. Generally, because an antibody has a bivalent binding capacity to an antigen (i.e., an antibody of 1 molecule has two antigen binding sites), the binding tendency to an antigen is strong and the half-life of a probe-target complex between an antibody and an antigen far exceeds the upper limit of the above scope. Thus, it is preferable that a fragment of an antibody that can repeatedly bind to and dissociate from a target substance be used and that a fragment of an antibody have a univalent binding capacity to a target substance, which is an antigen (i.e., 1 molecule has 1 antigen binding site). Examples of an antibody fragment having a univalent binding capacity to a target substance may include a Fab fragment, a Fab′ fragment, an scFv fragment, a VHH fragment of a single domain antibody (for example a commercial name of nanobody), etc.

Methods of producing the above antibody and fragment of an antibody are not particularly limited. A preferable embodiment for a method of screening for an antibody presenting the above prescribed half-life from candidates for antibodies adjusted by an arbitrary method such as a hybridoma method etc. will be explained later.

Luminescent substances contained in probes are not particularly limited as long as they are substances emitting light that makes observation possible under a prescribed condition. As a luminescent substance, a fluorescent substance that can emit fluorescence when irradiated with excitation light is particularly preferable. Examples of fluorescent substances may include a fluorescent protein such as a green fluorescent protein (GFP), an enhanced green fluorescent protein (EGFP), a red fluorescent protein (RFP), TagRFP, etc.; fluorescent dye such as Atto (trademark) 488, Atto (trademark) 550, Dylight (trademark) 488, Dylight (trademark) 550, CF (trademark) dye (CF680R, CF488A, CF543, etc.), etc.; and a quantum dot.

In an imaging step of the present invention, a mixture of a medium, a probe and an observation target sample is preferably exposed at intervals that are sufficiently shorter than a period of time between when the luminescent substance starts to be exposed to the prescribed condition and when it photobleaches the light (photobleaching period of time) or may be exposed consecutively, in the prescribed condition (irradiation with excitation light for example) for making a luminescent substance included in the probe emit light. For example, in an embodiment in which a luminescent substance is a fluorescent substance, a mixture of a medium, a probe and an observation target sample may be irradiated with excitation light at intervals that are sufficiently shorter than a photobleaching period of time or may be irradiated consecutively.

A luminescent substance in a probe and a binding substance can be linked through chemical binding via an appropriate linker component as necessary. Examples of chemical binding may include covalent binding, coordinate binding, etc., and covalent binding is preferable in view of stability. When a luminescent substance and a binding substance are both polypeptides, a probe can be formed as a fused polypeptide resulting from the luminescent substance and the binding substance linking to each other through normal peptide binding.

An imaging step of the present invention is performed in a state in which a medium containing a probe and a sample are in contact with each other. Media are not particularly limited as long as they allow a probe and a target substance to maintain the above binding characteristics, however they are usually liquid, and the liquid is preferably an aqueous solution, is more preferably an aqueous buffer solution adjusted to an appropriate pH, is more preferably an aqueous buffer solution adjusted to a pH of 6.1 to pH 7.5, and more preferably contains active oxygen remover. As an active oxygen remover, at least one type or two or more types selected from glucose oxidase, catalase, 2-mercaptoethanol, glucose, etc. can be used. An example of a proper amount of glucose oxidase may be 200 μg/ml, an example of a proper amount of catalase may be 35 μg/ml, an example of a proper amount of 2-mercaptoethanol may be 0.5%, and an example of a proper amount of glucose may be 4.5 mg/ml. As a buffer component for preparing an aqueous buffer solution, HEPES, Tris, etc. can be used.

It is preferable that the concentration of probes in a medium have appropriately been adjusted to a concentration that allows identification of emission of light of a luminescent substance as a separate speckle for each molecule in one speckle image.

Temperature conditions in a case when a medium containing a probe is brought into contact with a sample are not particularly limited, and for example 20 degrees Celsius through 30 degrees, and more preferably an ambient temperature, specifically 25 degrees Celsius, can be adopted.

4. Embodiment of Observation Method of Present Invention that Uses Microscope

Devices used for an observation method of the present invention are not particularly limited.

FIG. 4 shows an example of a device that can be used for an embodiment of the present invention in which a luminescent substance contained in a probe is a fluorescent substance that emits fluorescence when irradiated with excitation light.

A microscope apparatus 1 shown in FIG. 4 includes excitation illumination devices 10 (including a first excitation illumination device 11 and a second excitation illumination device 12), a microscope body 20, a camera 30, a camera controller 40, a control unit 50, a display device 60 and a storage unit 70.

The excitation illumination devices 10 (including the first excitation illumination device 11 and the second excitation illumination device 12) are devices that provide the microscope body 20 with excitation light for making a fluorescent substance emit light. Any device can be used as the excitation illumination devices 10 (including the first excitation illumination device 11 and the second excitation illumination device 12) as long as it can provide excitation light of a wavelength in accordance with the fluorescent substance. Although they are not shown, each of the first excitation illumination device 11 and the second excitation illumination device 12 may be a system combining a plurality of devices and usually includes a laser beam source, a shutter, and a total reflection mirror. The laser beam source is a light source that emits excitation light. The shutter is a device that switches between supply and suspend of excitation light to the microscope body 20. The total reflection mirror is a mechanism for totally reflecting excitation light emitted from the laser beam source toward a stage 21 of the microscope body 20.

The microscope body 20 may be an inverted microscope for example. The microscope body 20 includes the stage 21 for mounting thereon a sample that is to be observed. To the microscope body 20, the camera 30 for picking up a fluorescence image of the sample mounted on the stage 21 is connected. As the camera 30, for example a CCD camera having a plurality of pixels can be used.

The microscope body 20 is provided with a an objective lens for issuing excitation light toward the stage 21, an imaging lens for condensing, on the light receiving plane of the camera 30, fluorescence radiated from a fluorescent substance in the sample, etc. The above objective lens and the imaging lens constitute an imaging optical system.

The microscope body 20 including the stage 21 and the imaging optical system is configured to be able to provide total internal reflection illumination by which excitation light is totally reflected on the interface between the glass in contact with the sample and the sample. The total internal reflection illumination makes it possible to illuminate the sample with evanescent light that leaks to the sample side from the glass when the excitation light is totally reflected. In the shown embodiment, the second excitation illumination device 12 is equivalent to a device for providing excitation light through total internal reflection illumination.

The microscope body 20 is further configured to be able to switch between the above total internal reflection illumination and epi-illumination to use them. In the shown embodiment, the first excitation illumination device 11 is equivalent to a device for providing excitation light based on epi-illumination. Using a speckle image of fluorescence based on the total internal reflection illumination of excitation light and a speckle image of fluorescence based on epi-illumination makes it possible to calculate the position in the depth direction (z direction) in an observation image. Alternatively, it is also possible to make a probe emit intensive fluorescence by issuing excitation light based on epi-illumination and excitation light based on total internal reflection illumination simultaneously.

The excitation illumination devices 10 do not always have to include the first excitation illumination device 11 and the second excitation illumination device 12 and may have only one of the first and second excitation illumination devices 11 and 12.

The control unit 50 is a computer that totally controls the microscope apparatus 1, and is connected to the excitation illumination devices 10 (first excitation illumination device 11 and second excitation illumination device 12), the display unit 60, the storage unit 70, and the camera controller 40. The control unit 50 has at least a control signal generation function of generating a control signal for controlling these devices, a speckle image obtainment function of obtaining a speckle image via the camera controller 40, an image analysis function of analyzing an obtained speckle image, a light source control function of controlling driving of a light source included in the excitation illumination devices 10, and an image forming function of generating an observation image from a plurality of speckle images. The control unit 50 constitutes an image generation unit that generates an observation image as the overall function.

The camera controller 40 performs driving control of the camera 30. The camera controller 40 operates the camera 30 on the basis of a control signal input from the control unit 50 so as to obtain a speckle image of fluorescence, and outputs the obtained speckle image to the control unit 50.

The display unit 60 is a display (display device), a printer (printing device), etc., and provides a function of displaying and printing an image based on image data output from the control unit 50 (data of a speckle image, data of an observation image, etc.).

The storage unit 70 can be configured by a storage device such as a semiconductor memory, a hard disk etc. A program used in the control unit 50 and data provided from the control unit 50 (such as a speckle image etc.) are stored in a state in which they can be read by the control unit 50.

Hereinafter, explanations will be given for the observation method of the present invention, by the microscope apparatus 1, that uses a fluorescent substance emitting fluorescence when irradiated with excitation light as a luminescent substance contained in a probe.

An imaging step using the microscope apparatus 1 is a step in which a step is performed a plurality of times at different times respectively, where a sample that has been brought into contact with a medium containing a probe on the stage 21 is irradiated with excitation light by using the excitation light illumination devices 10 (one or both of the first excitation illumination device 11 and the second excitation illumination device 12) and a speckle image including, as a speckle of fluorescence, fluorescence emitted from a substance contained in a probe that has bound to a target substance in the sample is obtained by the camera 30, and thereby a plurality of speckle images are obtained. A step of obtaining each speckle image will be referred to as a “frame imaging step”. In each frame imaging step, the camera 30 picks up a speckle image of fluorescence emitted from a fluorescent substance each time excitation light is issued. A speckle image has a different pattern for each frame.

In each frame imaging step, the control unit 50 operates the camera 30 via the camera controller 40 so as to pick up an image of fluorescence emitted from a fluorescent substance, and thereby obtains a speckle image. An obtained speckle image is output to the control unit 50 from the camera controller 40. The control unit 50 also stores a thus-obtained speckle image in the storage unit 70.

In an imaging step, the control unit 50 repeatedly performs the above frame imaging step at appropriate intervals. Examples of the number of times of the frame imaging step may include hundreds of times through hundreds of thousands of times including for example 1,000 to 999,000 times.

An observation image generation step in the present invention is a step in which an observation image of a target substance that binds to the probe in the sample is generated from a plurality of speckle images obtained in the above imaging step. Specifically, the control unit 50 executes an appropriate computer program so as to obtain, for each of the plurality of speckle images recorded in the storage unit 70, information of position of a speckle included in a speckle image so as to generate an observation image by integrating the pieces of information from the plurality of speckle images. In this example, information of the position of each speckle is typically information of the central position (position of the center of gravity) of each speckle, and can be obtained by using for example DAOSTORM, a computer program (Nature methods, 8 279-280, 2011). In the above, in order to increase the accuracy, only a speckle of luminance that is equal to or higher than a prescribed threshold may be used for generating an observation image in each of the speckle images. When generating an observation image, an observation image can be generated by drawing, at the central position of each speckle of a blank image, a point having an appropriate size. Specifically, by turning the size of pixels of a blank image into an appropriate size (for example a square pixel with the length of each side being 5 nm through 20 nm) and by plotting the central position of each speckle to each pixel, an observation image can be generated. The control unit 50 records the data of the generated observation image in the storage unit 70 and also displays it in the display unit 60.

When a plurality of target substances exist in one sample, it is sufficient to perform the above imaging step sequentially by using probes that are specific to the respective target substances. “Probes that are specific to respective target substances” refer to probes that repeatedly bind to and dissociate from the respective target substances directly and specifically. It is preferable that the sample be washed sufficiently between the respective imaging steps. Then, the control unit 50 can generate an observation image of each target substance in the sample from a plurality of speckle images, stored in the storage unit 70, obtained in the respective imaging steps. The control unit 50 can also synthesize pieces of data of observation images of a plurality of target substances in the same sample so as to output the result to the display unit 60 and display a multiple-observation image resulting from superposing observation images of a plurality of target substances.

The above is an embodiment of the observation method of the present invention that uses a probe containing a fluorescent substance as a luminescent substance. When a luminescent substance is a luminescent substance that is not a fluorescent substance, the method of the present invention can be implemented through the same procedures as that described above if means for providing a condition in a sample mounted on the stage 21 for that luminescent substance to radiate light instead of the excitation illumination devices 10 in the microscope apparatus 1 are arranged and the camera 30 that can pick up an image of light emitted by the luminescent substance in the probe that has bound to a target substance in the sample as a speckle is used.

5. Probe and Kit

The present invention also provides the above probe itself and a kit including at least the above probe.

The above probe may be provided solely in solid form such as powder, may be provided in liquid form that has dispersed or has been dissolved in an appropriate liquid medium or may be provided in solid form such as powder together with an appropriate solid component (such as a diluting agent etc.). In other words, a probe of the present invention may be provided solely by itself or may be provided as a probe-containing composition that includes at least a probe and may include a different complementary component.

Any kit may be used as the above kit as long as it includes at least a probe of the above various forms and it may further include a different element used for observing a target substance. Examples of a different element for the above kit may include a medium such as a liquid medium for dissolving or dispersing the probe etc., and a reagent to be used for a process of observing a sample etc. The above medium may be a medium that includes a probe and that is used for bringing it into contact with a sample for performing observation. The probe and a different element in the above kit are usually wrapped separately so that they will not be mixed physically.

6. Screening Method

The present invention also provides a screening method of a site (also referred to as a target substance identification site or a binding substance) in which a target substance is identified in the probe accordingly, the screening method including:

-   -   an immobilization step in which a candidate substance of the         site or a substance partially containing the candidate substance         is fixed to a solid support;     -   an observation step in which a target substance linked to a         luminescent substance and a solid support obtained in the         immobilization step are observed in a medium while the target         substance linked to a luminescent substance and the solid         support obtained in the immobilization step are kept in contact,         in a condition that allows observation, in units of 1 molecule,         of light emission from the luminescent substance in a         probe-target complex formed by binding between the target         substance and the candidate substance, and     -   a screening step in which the candidate substance resulting in a         half-life of the probe-target complex that is equal to or more         than 10 milliseconds and equal to or less than 3 seconds is         selected as the site on the basis of observation in the         observation step.

According to this embodiment of the present invention, it is possible to efficiently perform screening for a substance that presents a desired binding tendency to a target substance from a library of candidate substances.

Examples of a solid support used for an immobilization step may include an inner wall surface of each well of for example a multiwell plate.

Means for fixing a candidate substance to a solid support in an immobilization step are not particularly limited, and when for example a candidate substance is an antibody or an antibody fragment, it is preferable that a Fab domain of an antibody or an antibody fragment be immobilized to the solid support in a state in which it can bind to the target substance. For example, by fixing to a solid support a protein having a binding capability to an Fc domain of an antibody (for example, Protein G) and then immobilizing the antibody to the solid support to which the protein has been immobilized, it is possible to immobilize the antibody to the solid support in a state in which the Fab domain can bind to the target substance. Methods of immobilizing the protein to a solid support are not particularly limited, however it is possible to immobilize the protein via a functional group introduced to a surface of a solid support.

As a medium used in an observation step, the same medium as that used in an imaging step of the present invention can be used.

Types of luminescent substances are not particularly limited in a target substance linked to the luminescent substance used in an observation step, however a fluorescent substance that can emit fluorescence when irradiated with excitation light is particularly preferable. Specific examples of a fluorescent substance are the same as those of the fluorescent substance described above for a probe.

In an observation step, in a medium, a target substance linked to a luminescent substance and a solid support to which a candidate substance is fixed are observed in a state in which they are in contact with each other, in a condition that allows observation, in units of 1 molecule, of emission of light by the luminescent substance in a probe-target complex of the target substance and the candidate substance. In this step, the fact that light emission of a target substance that has not bound cannot be detected because it causes thermal motions randomly in the medium, whereas emission of light from the target substance that has bound to the candidate substance can be detected and is utilized. In order to observe emission of light by the luminescent substance in units of 1 molecule, it is effective to reduce the concentration of the target substance in the medium. Even when a candidate substance is a bivalent antibody or antibody fragment having two antigen binding sites, reducing the concentration of the target substance in the medium makes it possible to observe binding of the target substance individually in each antigen binding site.

Observation means in an observation step may appropriately be selected in accordance with the luminescent substance. When the luminescent substance is a fluorescent substance, observation is possible by using a fluorescence microscope (for example, a TIRF fluorescence microscope).

In a screening step, on the basis of observation in the observation step, the candidate substance leading to the half-life of the probe-target complex that is equal to or more than 10 milliseconds and equal to or less than 3 seconds is selected as a target substance identification site. The half-life is more preferably equal to or more than 10 milliseconds and equal to or less than 2 seconds, more preferably equal to or more than 10 milliseconds and equal to or less than 1 second, more preferably equal to or more than 10 milliseconds and equal to or less than 900 milliseconds, more preferably equal to or more than 10 milliseconds and equal to or less than 800 milliseconds, more preferably equal to or more than 10 milliseconds and equal to or less than 700 milliseconds, more preferably equal to or more than 10 milliseconds and equal to or less than 600 milliseconds, more preferably equal to or more than 10 milliseconds and equal to or less than 500 milliseconds, more preferably equal to or more than 20 milliseconds and equal to or less than 300 milliseconds, and particularly preferably equal to or less than 250 milliseconds. The half-life is defined by a period of time before the number of target substances that have bound to candidate substances on solid supports at a given moment is reduced to half through dissociation. Measurement procedures for the half-life are as below. A medium containing a target substance labeled with a luminescent substance is brought into contact with a solid support to which the candidate substance has been fixed, and a period of time between when a light emission speckle appears and when it disappears is measured, for each light emission speckle, while performing observation under a condition used for observation. Then, periods of time between the appearance and disappearance of the speckles are plotted in accordance with a complementary cumulative relative frequency function (1-Ndissociation). Then, by fitting the above complementary cumulative relative frequency with an exponent function, that half-life is calculated.

A period of time between appearance and disappearance of a light emission speckle based on a labeled target substance is measured by the following procedures. Specifically, while keeping a medium containing a labeled target substance in contact with a solid support to which the candidate substance has been immobilized and providing a prescribed condition necessary for emitting light (for example irradiation with excitation light), speckle images including speckles based on the light emission of the labeled target substance are consecutively picked up with an exposure time of X seconds (for example 0.050 seconds, 0.100 seconds, etc.) and at a frame rate of 1/XHz. Then, a period of time is measured between when a target substance that has bound to a candidate substance appears in a speckle image and when it disappears through dissociation. In the above, observation is performed for Y consecutive frames, and for a speckle not observed in frames before and after them, the period of time between the appearance and disappearance of that speckle can be treated as YX seconds.

In a screening method of the present invention, the candidate substance is more preferably an antibody or a fragment of an antibody that binds to a prescribed target substance and fixes the antibody to a solid support in the immobilization step. When screening is performed for antibodies binding to prescribed target substances, ELISA is generally used in which target substances are immobilized to solid supports and candidate antibodies are brought into contact with the solid supports. In a screening method according to the present embodiment, contrary to ELISA, an antibody (the candidate substance) is solid phased. Then, in the medium, an antigen (target substance) linked to a luminescent substance and the solid support are observed in a state in which they are in contact with each other, in a condition that allows observation, in units of 1 molecule, of emission of light by the luminescent substance in a probe-target complex formed by binding of the target substance and the candidate substance. This method has advantages as described below.

1) It is possible to measure the half-life of binding of an antibody and an antigen. Because binding/dissociation between an antibody and an antigen is visualized through the appearance and disappearance of a fluorescent single molecule, the half-life of that binding can be measured directly for each single molecule of an antigen.

2) It is possible to measure univalent binding/dissociation of an antigen and an antibody. Epitope that allows identification of a monoclonal antibody is at one location in an antigen. Accordingly, an antigen that has bound to 1 antigen binding site of a solid-phased antibody cannot bind to a different antigen binding site existing nearby, and an antigen inevitably binds with the antibody molecule in a univalent state. When an antigen has been solid phased, an antibody can bind to one or two antigens, making it impossible to measure univalent binding/dissociation of an antigen and an antibody. A method in which this antibody is solid phased makes it possible to estimate the half-life of binding of an antibody fragment such as a Fab fragment that binds in a univalent manner without producing it.

3) It is possible to perform screening for an antibody fragment from a library of hybridoma in an inexpensive and simple manner. In one embodiment of this screening method, main structure factors are a solid support resulting from immobilizing an antibody contained in each supernatant of a hybridoma library and a labeling body of antigen epitope. The half-life of binding of an antibody fragment and an antigen can be estimated even without producing a light-emission-labeled antibody fragment from an antibody of each supernatant of a library, making it possible to perform screening on candidate substances at a very low cost. Also, visualization is performed in units of molecules, measurement is possible even when the amount of antibodies that have been solid phased is small. Thereby, immense amounts of labor and time taken for cultivation of a hybridoma library can be saved.

The present screening method has solved a major problem in evaluation of the affinity of an antibody by ELISA. 1) In ELISA, an antigen is solid phased, and accordingly an antibody may bind to an antigen regardless of whether the binding is univalent or bivalent. Further, by a secondary antibody used for measuring a binding amount of primary antibodies, a primary antibody is crosslinked. This prevents measurement of the half-life of univalent binding of an antibody and an antigen. 2) The binding amount of antibodies obtained in ELISA is influenced not only by the affinity of antibodies but also by the amount of antibodies produced by hybridoma. This makes it difficult to evaluate the property of binding of one antibody in ELISA. 3) In addition, because washing is conducted a plurality of times in a reaction step in ELISA, there is a risk that an antibody that has bound with a short half-life that is equal to or less than 3 seconds, which is needed in the present invention, may be lost before being visualized. The method of the present embodiment has solved these problems by solid phasing an antibody instead of an antigen and visualizing binding/dissociation of an antigen and an antibody in actual time. The method of the present embodiment is more suitable for selecting an antibody having a short binding half-life, which is the aim of the present invention, than ELISA in principle in that it can evaluate univalent binding/dissociation, it is not influenced by the production amount of antibodies, and it can also measure a short binding half-life.

According to a screening method of the present invention, a library of polypeptides and a library of phage display can be solid phased as candidate substances for a target substance identification site of a probe in addition to a antibody or an antibody fragment. A screening method of the present invention can also be applied to solid-phasing of these for performing screening for a polypeptide that binds to and dissociates from a target substance.

The present invention will be explained in more detail by the following examples, however they are just exemplary and do not limit the present invention. All the experiments below were conducted at an ambient temperature, i.e., 25 degrees Celsius, unless limitations are particularly given.

1. Experiment 1 Methods Plasmid and Reagent

Expression plasmid (pFLAG-EGFP-C1) encoding EGFP (sequence number 1) having an N terminus tagged with FLAG (sequence number 2) and an expression plasmid (p3×FLAG-EGFP-N3) encoding EGFP having a C terminus tagged with 3×FLAG (sequence number 3) were constructed respectively by using a pEGFP-C1 vector and a pEGFP-N3 vector (Clontech Laboratories, Inc).

EST clones encoding mouse MAP4, human Tau isoforms 3 and 4, mouse KIF1A, human plectin-1 and Xenopus laevis talin-1 were purchased from OpenBiosystems.

cDNA encoding human FAK was purchased from the DNASU Plasmid Repository.

The GenBank/EMBL/DDBJ accession numbers for each sequence are as follows:

BC055332 (MAP4), BC114948 (Tau isoform 3), BC101936 (Tau isoform 4), BC062891 (KIF1A), BM559026 (Plectin-1), CF282569 (Talin1) and BC035404 (FAK).

cDNAs encoding human EB1, rat CLIP-170, human CLASP2γ and human APC were provided by Y. Mimori-Kiyosue (RIKEN).

As plasmids encoding human paxillin, chicken Src and human vinculin, those described in documents 31 to 33 were used.

Each cDNA was inserted into a pFLAG-EGFP-C1 vector or a p3×FLAG-EGFP-N3 vector by using a PCR. For a probe having an N terminus at the fusion position of GFP in table 1 below, a pFLAG-EGFP-C1 vector was used. For a probe having a C terminus at the fusion position of GFP in table 1 below, a p3×FLAG-EGFP-N3 vector was used.

An expression plasmid encoding PIPKIγ fragment (amino acid residues 641-668) having an N terminus tagged with FLAG-EGFP was constructed by inserting a synthetic cDNA encoding PTDERSWVYSPLHYSAQAPPASDGESDT (sequence number 18) into a pFLAG-EGFP-C1 vector.

Lifeact peptide (MGVADLIKKFESISKEE (sequence number 19)) with an Atto 488 fluorescent body linked to an N terminus was purchased from Sigma-Aldrich.

The amino acid sequence of EGFP is denoted by sequence number 1.

The amino acid sequence of FLAG is denoted by sequence number 2.

The amino acid sequence of 3×FLAG is denoted by sequence number 3.

The amino acid sequence of mouse MAP4 is denoted by sequence number 4.

The amino acid sequence of human Tau isoform 3 is denoted by sequence number 5.

The amino acid sequence of human Tau isoform 4 is denoted by sequence number 6.

The amino acid sequence of mouse KIF1A is denoted by sequence number 7.

The amino acid sequence of human Plectin-1 is denoted by sequence number 8.

The amino acid sequence of Xenopus laevis Talin1 (amino acid residues 1-2353) is denoted by sequence number 9.

The amino acid sequence of human FAK is denoted by sequence number 10.

The amino acid sequence of human EB1 is denoted by sequence number 11.

The amino acid sequence of rat CLIP-170 is denoted by sequence number 12.

The amino acid sequence of human CLASP2γ is denoted by sequence number 13.

The amino acid sequence of human APC is denoted by sequence number 14.

The amino acid sequence of human Paxillin is denoted by sequence number 15.

The amino acid sequence of chicken Src is denoted by sequence number 16.

The amino acid sequence of human Vinculin is denoted by sequence number 17.

The amino acid sequence of human PIPKIγ-90 fragment (amino acid residues 641-668) is denoted by sequence number 18.

The amino acid sequence of Lifeact peptide is denoted by sequence number 19.

Production and Screening of Exchangeable Protein Probes

In order to find exchangeable probes for super-resolution images of microtubules, intermediate filaments and focal adhesions, a test was conducted by using, as candidate molecules for probes, a protein (polypeptide) and a protein fragment that are known to be able to localize in each target structure. “Exchangeable” used herein refers to being able to repeatedly bind to and dissociate from a target structure.

Table 1 shows the tested probe candidates. The test was conducted a plurality of times by using, as a probe candidate, a protein or a fragment of a protein mentioned in the section of “plasmid and reagent”.

An expression plasmid for expression as a molecule made to fuse with EGFP was constructed for each probe candidate in accordance with the documents described in table 1.

HEK (Human embryonic kidney)-293F cells were transfected with a plasmid encoding a probe candidate protein tagged with FLAG-EGFP or 3×FLAG-EGFP. 3 to 4 days later, the cells were dissolved in a cell lysis buffer (10 mM HEPES, pH 7.2, 90 mM KCl, 3 mM MgCl2, 0.2% Triton X-100, 100 μM DTT) containing a protease inhibitor cocktail (Nacalai Tesque). Centrifugal separation was performed on the lysate and the supernatant liquid was collected. In order to perform screening for a probe for IRIS, the binding capacity of a probe candidate in the supernatant liquid with respect to the structure of an XCT cell that was fixed with paraformaldehyde (PFA) and that received a permeabilization process was tested. The appearance and disappearance of single molecule speckles (Single-molecule speckles, SiMS) in the structure was tested. Screening was performed for a probe for IRIS in accordance with the following criteria:

(1) It is possible to confirm a distribution of target substances in the structure in an image resulting from adding SiMS images; (2) It is possible to wash and remove a probe after SiMS imaging (imaging step); (3) A probe that has bound can dissociate from the target substance swiftly (half-life is equal to or less than 500 ms); and (4) It is possible to reconstruct an image of a target substance by integrating the central positions of the respective speckles.

Table 1 describes that “localization” is positive (P) when above criterion (1) is met, that “washability” is positive (P) when above criterion (2) is met, and that “IRIS image” is positive (P) when above criterion (3) is met.

Probes used for an IRIS experiment were purified in the following procedures. Specifically, each probe was overexpressed in HEK-293F cells and collected with anti-DYKDDDDK (Flag) antibody beads (Wako). The beads were washed four times with an excess amount of HEPES-buffered solution (10 mM Hepes pH 7.2, 90 mM KCl, 3 mM MgCl2, 100 μM DTT). Proteins that had bound to beads after the washing were processed with the HEPES-buffered solution containing 0.5 mg/ml DYKDDDDK (Flag) peptide (Wako) or 3×FLAG peptide (Sigma-Aldrich) for 30 minutes and were eluted.

The localization test of the above (1), whose result is shown in table 1, was performed in the following procedures. First, the supernatant liquid of lysate of a cell that expressed each probe candidate was brought into contact with an XCT cell that was fixed on a coverglass and that received a permeabilization process. A coverglass on which the sample was mounted was arranged in the observation chamber of a fluorescence microscope apparatus, which will be described in detail in the section of “procedures for imaging of multicolor super-resolution by IRIS” below, and speckle images of 10000 frames were obtained with an exposure time of 50 ms or 100 ms for one frame and at a frame rate of 20 Hz (20 frames per second) or with an exposure time of 100 ms and at a frame rate of 10 Hz (10 frames per second) while irradiating the sample with a 488 nm-laser beam line (with a main body output of 50 mW but reaching the sample after being attenuated by AOTF etc.) for total internal reflection fluorescence observation. The obtained speckle images of 10000 frames were integrated so as to confirm whether or not a distribution of target substances in the cell (microtubule, intermediate filament, focal adhesion or actin filament) was able to be confirmed. When a distribution of a target substances was able to be confirmed, the result of the localization test was treated as P (positive).

The localization test of the above (2), whose result is shown in table 1, was performed in the following procedures. After the picking up of speckle images explained in the previous paragraph, 1 ml of the supernatant liquid containing respective probe candidates in the observation chamber was aspirated by using an aspirator and 1 ml of an imaging solution (however, it was not supplemented with active oxygen-scavenging mix) that does not contain a probe and that will be described below was added. This switching of imaging solution was conducted slowly so that the observation position would not shift. Next, an imaging solution not containing a probe and not containing the active oxygen-scavenging mix was switched 10 to 20 times. After the switching, for confirmation, for an XCT cell sample in the observation chamber, speckle images of 10 frames were picked up under a similar condition to that of the localization test described in the previous paragraph, and when the number of speckles confirmed in the speckle images (i.e., the number of probes that had bound) became sufficiently smaller (equal to or less than about 10%) than the number of the speckles in the speckle images before the washing operation, washing was determined to be possible and the result was treated as P (positive).

The measurement of binding half-life, whose result is shown in table 1, was performed in the following procedures. Speckle images of 10000 frames picked up in the localization test were used in order to measure a period of time between the appearance of a probe that has bound to a target in a speckle image and disappearance through dissociation in a semi-manual mode by using Speckle TrackerJ, an ImageJ plug-in. In case of for example picking up of images with an exposure time of 50 ms and at a frame rate of 20 Hz, it is assumed that a period of time between the appearance and disappearance of a speckle that is observed in only one of consecutive frames is 50 ms and a period of time between the appearance and disappearance of a speckle observed in only two consecutive frames is 100 ms. Then, the number of binding probes with respect to periods of time between the appearance and disappearance was plotted in accordance with a complementary cumulative relative frequency function (1-Ndissociation). Ndissociation is a cumulative relative frequency of probes that dissociated. Then, by fitting the complementary cumulative relative frequency function with an exponent function, the half-life was calculated. However, the binding half-life with respect to an actin filament of Atto488-Lifeact was measured by using a method that is described in detail in the section for FIG. 9 in the Brief Description of the Drawings.

The IRIS image test of the above (3) whose result is described in table 1 was performed in the following procedures. By using all the speckle images of 10000 frames picked up in the localization test, the central point of a probe that has bound to a target in each frame of the speckle images was determined with nanometer accuracy by using DAOSTORM. By adding a large number of pieces of central point information in the fluorescence images of 10000 frames, a reconstruction image (observation image) was generated. Whether or not a distribution of a target molecule was able to be observed in that reconstruction image was determined, and when a distribution of a target molecule was observed, the result was treated as P (positive).

Procedures for Imaging of Multicolor Super-Resolution by IRIS

Xenopus laevis XTC cells were cultured in 70% Leibovitz's L15 medium supplemented with 10% fetal bovine serum. A multicolor super-resolution image was produced from a large number of fluorescence single molecule speckle (SiMS) images that were sequentially obtained from a fixed and XTC cell (20,000 to 500,000 frames per probe) with various exchangeable probes. The cells were allowed to spread on a 0.1 mg/ml poly (L-lysine) and 10 g/ml fibronectin-coated coverglass in 70% Leibovitz's L15 medium without serum and distinct stress fibers and focal adhesions were formed (document 33). 2 hours later, the cells were fixed and received a permeabilization process with a cytoskeleton buffer containing 3.7% PFA and 0.5% Triton X-100 in (10 mM Mes pH6.1, 90 mM KCl, 3 mM MgCl2, 2 mM glycol ether diamine tetraacetic acid (EGTA)). After performing blocking with 4% bovine serum albumin for 30 minutes, the purified IRIS probes were brought into contact with the cells in an imaging solution including the HEPES-buffered solution (10 mM Hepes pH 7.2, 90 mM KCl, 3 mM MgCl2, 100 μM DTT) with an oxygen-scavenging mix (200 μg/ml glucose oxidase, 35 μg/ml catalase, 4.5 mg/ml glucose, 0.5% 2-mercaptoethanol) (document 34). The concentration of the probe was 1 nM through 100 nM. When the oxygen-scavenging mix was not used, laser-induced photodamage was apparent after obtaining SiMS images several tens of thousands of times.

For the imaging of actin filaments in vitro, monomeric actin was prepared from rabbit skeletal muscle in a method described in documents 33, 35 and 36. Phalloidin-stabilized F-actin was observed on a 1 mg/ml poly (L-lysine)-coated coverglass in the imaging solution.

SiMS images were obtained by using an inverted microscope (Olympus IX83-ZDC) equipped with an Olympus PlanApo 1.45×100 through a numerical aperture (NA) objective lens, a 2×intermediate lens and an EM-CCD camera (Evolve 512, Roper), and controlled by MetaMorph software (Molecular Device). The focus was automatically maintained at the bottom of the cell by a z drift compensation system during the long-term imaging. The IRIS probe was alternately excited with a 473-nm laser beam (50 mW) for epi-illumination microscopy and a 488-nm laser beam (50 mW) for epi-fluorescence observation in the following procedures. In the epi-illumination mode, the incidence angle of the 473-nm laser beam was tilted so as to reduce background fluorescence from out-of-focus probes that have not bound. The epi-fluorescence image and TIRF image were used to estimate the z position of the target object (see below). Specifically, images were picked up by repeating the following procedures:

(a) imaging in bright field; (b) imaging of a SiMS image (speckle image) with epi-fluorescence (exposure time for one frame: 50 ms, frame rate: 20 Hz (20 frames per second), number of frames picked up consecutively: 250 frames); and (c) imaging of SiMS image (speckle image) with TIRF (exposure time for one frame: 50 ms, frame rate: 20 Hz (20 frames per second), number of frames picked up consecutively: 250 frames).

An image obtained in a bright field was used to correct a drift of the microscope stage in a lateral direction (see below) In case of observation of focal adhesions, epi-fluorescence observation was not performed and TIRF observation was performed (frame rate: 20 Hz, 500 frames). Each one of the procedures required 27 seconds and was repeated 160 to 240 times (probe/target substance=CLIP-170 fragment) (amino acid residues 3-309 of sequence number 12)/microtubule, 40 times (probe/target substance=PIPKIγ fragment (641-668) (sequence number 18), Paxillin (overall length of sequence number 15) and the Src fragment (amino acid residues 3-251 of sequence number 16)/focal adhesion), 800 times (probe/target substance=Lifeact (sequence number 19)/actin filament), and 400 to 480 times (probe/target substance=Plectin-1 fragment (amino acid residues 4022-4364 of sequence number 8/intermediate filament). In order to maintain the oxygen-scavenging capacity in the imaging solution, an imaging solution containing the probe was replaced with a fresh imaging solution every 40 sets when the CLIP-170 fragment, the PIPKIγ fragment, paxillin and the Src fragment were used and every 80 sets when Lifeact and the plectin-1 fragment were used. For multicolor imaging of three types of cytoskeletons and focal adhesions, a pick up of SiMS images was conducted in the order of the CLIP-170 fragment, the PIPKIγ fragment (or the Src fragment and paxillin), Lifeact and the plectin-1 fragment. After obtaining SiMS images on the basis of each probe, washing was conducted 10 times by using the HEPES-buffered solution. The remaining fluorescence of a probe was completely photobleached in the HEPES-buffered solution supplemented with an oxygen-scavenging mix, and the next probe was made to react.

In the present example, imaging steps of obtaining observation images were conducted under the above conditions unless otherwise described.

Procedures for Image Reconstruction in IRIS

A super-resolution image was reconstructed by plotting the central points of each fluorescent speckle on a blank image with subpixel accuracy. The number of plotted points was typically 10⁶ to 10⁸. A central point was estimated with subpixel accuracy by fitting of a point-spread function (PSF) of this microscope using a computer program known as DAOSTORM (document 14). In order to correct a stage drift of the microscope, the drift distance was calculated by an autocorrelation function, i.e., A_(N) (x_(drift), y_(drift)) of the bright-field images obtained at each set of imaging procedures.

$\begin{matrix} {{A_{N}\left( {x_{drift},y_{drift}} \right)} = {\sum\limits_{y = y_{0}}^{y_{m}}{\sum\limits_{x = x_{0}}^{x_{m}}\left\lbrack {{I_{0}\left( {x,y} \right)} \times {I_{N}\left( {{x + x_{drift}},{y + y_{drift}}} \right)}} \right\rbrack}}} & \left\lbrack {{Numerical}\mspace{14mu} {expression}\mspace{14mu} 1} \right\rbrack \end{matrix}$

where x_(drift) and y_(drift) are drift distances in the directions of the x axis and y axis, respectively. I₀ (x, y) and IN (x+x_(drift), y+y_(drift)) are intensities at the pixel positions (x, y) and (x+x_(drift), y+y_(drift)) in the bright field images obtained in the 1st and Nth sets, respectively.

The product of I₀ (x, y) and IN (x+x_(drift), y+y_(drift)) is integrated within a prescribed region in the bright-field image. A_(N) (x_(drift), y_(drift)), which is a function of variables x_(drift) and y_(drift), becomes the maximum when the two bright-field images coincide. The x_(drift) and y_(drift) values leading to a maximum value as A_(N) (x_(drift), y_(drift)) were calculated by using a customized plug-in in ImageJ software (http://rsb.info.nih.gov/ij/). First, the drift of the bright-field image in the Nth set was corrected using the x_(drift) and y_(drift) values with pixel accuracy. To further determine the drift distance with subpixel accuracy, the bright-field image and the corrected image in the first and Nth sets were enlarged by using a bicubic method. Using the A_(N) (x_(drift), y_(drift)) of the enlarged images, the drift distances were determined with subpixel accuracy. The central positions of speckles in the SiMS images in the Nth set were corrected with the drift distances. By plotting the corrected central positions, a super-resolution image was produced. The positions of speckles consecutively observed in 10 or more frames or in 20 or more frames were not used when generating a reconstruction image (observation image) based on Lifeact or generating a reconstruction image (observation image) based on the plectin-1 fragment. These two probes in some cases bound to a target substance when strong excitation laser output was employed.

Image Process for Mapping z Position of Observation Target Object

Because a TIRF excitation light intensity exponentially decreases with increasing distance from the coverglass, the height of the observation target object was estimated from the ratio of a TIRF image and an epi-fluorescence image. The z position from the coverglass surface was measured by the method, described in document 18, using fluorescent microtubules that were tilted with respect to the coverglass in a low-melting-point agarose gel. HyLight 488-labeled tubulin was purchased from Cytoskeleton. The labeled microtubules were prepared according to the method described in document 18. Images of the tilted microtubules each having one end touching the coverglass were picked up by TIRF and epi-fluorescence. Epi-fluorescence images were obtained as z stack images (0.2-μm step size) (FIG. 14 (a)). In the intensity line profile along the microtubules of epi-fluorescence images, the x-y position of the highest intensity was used to determine the intersection of the tilted microtubule and the focal plane (FIG. 14 (a) arrows). By connecting the intersections among the z-stacked images, the z-directional distance of each point along the tilted microtubule was obtained. The z profile of the excitation light intensity based on a TIRF method was determined by associating the z-directional distance of each point with the ratio of the intensity of the microtubule in a TIRF image and the intensity of the microtubule in an epi-fluorescence image (FIG. 14 (b)). This z profile was fitted with a single exponential decay function (FIG. 14 (b)). The scale of the z positions were again adjusted by a factor of 0.82, taking into consideration the difference in the refractive index between the immersion oil and the imaging solution (document 18). The inverse function of the exponential function was used to determine the z position of the observation target object by the following numerical expression.

$\begin{matrix} {z = {{- \alpha_{z}}{\ln \left( {\beta \frac{F^{TIRF}}{F^{Epi}}} \right)}}} & \left\lbrack {{Numerical}\mspace{14mu} {expression}\mspace{14mu} 2} \right\rbrack \end{matrix}$

(where α_(z) is the z position at which the intensity of TIRF illumination is 1/e, β is a parameter for calibrating the difference in the laser output between TIRF observation and epi-fluorescence observation, and F^(TIRF) and F^(Epi) are the fluorescence intensities of the target object in a TIRF image and an epi-fluorescence image, respectively)

The z-position maps of three types of cytoskeletons were converted from the image of a ratio of the IRIS image (observation image) based on TIRF to the IRIS image (observation image) based on an epi-fluorescence image. For this purpose, the peak intensity of the fluorescent speckle was also fitted by using DAOSTORM. The IRIS images were reconstructed by plotting the peak intensity at the central position of the speckle. In the obtained z-position map, the fluorescence intensity in an image resulting from adding the IRIS image based on TIRF and the IRIS image based on epi-fluorescence was masked by a threshold and noise in a region having no cytoskeletons was removed. In a layered structure such as an actin stress fiber, etc., the calculated z position represents the height of the center of gravity of the structure in the z axial direction.

The z position of the microtubule plus end traced by live-cell imaging of EB1-EGFP was obtained by converting a ratio of an average intensity in a 0.4-μm-diameter region of the microtubule plus end in the TIRF image to that in a corresponding region in the epi-fluorescence image.

Live-Cell Imaging of the Movement of Microtubule Plus End

XTC cells were transfected with an expression plasmid of EGFP fused EB1. 3 to 4 days later, live-cell imaging of EB1-EGFP was conducted on the cells at one-second intervals. In each interval term, an imaging with an exposure time of 100 ms of fluorescence by excitation light for epi-fluorescence observation and imaging with an exposure time of 100 ms of fluorescence by excitation light for total reflection fluorescence observation were conducted. In the above, each excitation light was emitted under the above conditions for IRIS super-resolution except that the excitation light was emitted after having its laser power reduced to about 20% of that used for an IRIS super-resolution imaging in order to avoid damaging live cells. At each imaging time point, two fluorescence images were obtained by alternately using total internal reflection illumination and epi-illumination with an exposure time of 100 milliseconds. An EB1-labeled microtubule plus end was traced by using Speckle TrackerJ, an ImageJ plug-in (documents 33 and 37). The z position of the traced microtubule plus end was calculated by the above method. The speed of the site of the microtubule plus end was calculated with a linear approximation of its x-y positions at five consecutive imaging time points.

Results

The results of screening tests on probe candidates were as below.

TABLE 1 AMINO TARGET SEQUENCE ACID EGFP FUSION LOCALI- WASH- HALF- IRIS SUBSTANCE PROBE NUMBER NUMBER POSITION ZATION ABILITY LIFE IMAGE DOCUMENT MICROTUBULE CLIP-170 12  3-309 N TERMINUS P P 44 ms P 41 APC 14 2536-2843 C TERMINUS P P 100 ms P 43.44 APC 14 2781-2819 N TERMINUS P P 27 ms P 43.44 MAP4 4  1-908 N TERMINUS P P 109 ms P 47 MAP4 4 659-908 N TERMINUS P P 106 ms P 47 TAU ISOFORM 3 5  1-383 N TERMINUS P P 110 ms P 48.49 TAU ISOFORM 4 6  1-352 N TERMINUS P P 60 ms P 48.49 INTERMEDIATE Plectin-1 8 3777-4684 N TERMINUS P P 457 ms P 16 FILAMENT Plectin-1 8 3777-4364 N TERMINUS P P 59 ms P 16 Plectin-1 8 3777-4313 N TERMINUS P P 52 ms P 16 Plectin-1 8 4022-4364 N TERMINUS P P 103 ms P 16 Plectin-1 8 4066-4364 N TERMINUS P P 7.7 s NA 16 FOCAL Paxillin 15  1-557 N TERMINUS P P 196 ms P 50 ADHESION Paxillin 15  54-557 N TERMINUS P P 246 ms P 50 Paxillin 15  54-498 N TERMINUS P P 91 ms P 50 Paxillin 15 167-557 N TERMINUS P P 138 ms P 50 Src 16  1-251 C TERMINUS P P 161 ms P 59 Src 16  3-251 C TERMINUS P P 141 ms P 59 PIPKly90 18 641-668 N TERMINUS P P 496 ms P 17 ACTIN Atto488-Lifeact 19 — — — — 23 ms — — FILAMENT

In the table, P represents positive while NA indicates that measurement was not conducted (not accessed).

-   -   In the sequence table, sequence number 18 represents an amino         acid sequence of fragments 641-668 of PIPKIγ90.

A probe candidate that had not passed either a localization test or a washing test received neither a test of an IRIS image nor half-life measurement, and thus is not described in the above table 1. A probe candidate that did not pass a test is estimated to have a very long or very short binding half-life to a target substance or to not bind to a target substance.

Because the test described in detail in FIG. 9 and the explanations for the figure have independently confirmed that Atto488-Lifeact is effective for an actin filament and that the half-life thereof is 23 nm, these facts are described in the above table as reference results.

Super-Resolution of Actin Filament in High Labeling Density by IRIS

The present inventors confirmed whether or not a super-resolution image can be generated through the IRIS method by using Lifeact, which is a widely used actin marker. Lifeact is a short peptide that stains an actin filament in a live cell or a fixed cell (document 10). Lifeact has a property of being exchanged within 0.4 seconds on an actin filament (document 10). The high-speed exchangeability of Lifeact is confirmed through single-molecule speckle (SiMS) microscopy (documents 11 to 13). The dwell time of Atto488-labeled Lifeact showed a single exponential decay of a half-life of 23 milliseconds (FIG. 9 (a)) The present inventors selected a concentration of 2.4 nM as the Atto488-labeled Lifeact concentration in the imaging solution and obtained 2×10⁵ SiMS images of Lifeact on an actin filament in vitro through consecutive imaging using a total internal reflection illumination of 488 nm with an exposure time of 50 ms/frame and at a frame rate of 20 Hz. The central position of each fluorescent speckle was determined by using a computer program named DAOSTORM (document 14) (FIG. 9 (b)). Pieces of position information from a great number of Lifeact speckles in the above speckle images of 2×10⁵ frames were integrated so as to reconstruct an image of actin filaments (FIG. 5 (b) and FIG. 9 (c)). The average width of single actin filaments was 23 nm as the full-width at half-maximum (FWHM) in an image reconstructed by using only high-brightness speckles (highest 12% approximately) in order to guarantee a high accuracy of localization (FIG. 5 (c) and FIG. 9 (d)).

A major problem in conventional super-resolution microscopy is that using an antibody and a photoactivatable fluorescent protein makes it difficult to label an observation target structure in a sufficient density. The actin subunit and the antibody have the widths of 6 nm and 12 nm, respectively, and accordingly a single actin filament having 360 subunits per 1 μm can only be used for labeling up to a density of at most 180 subunits per 1 μm in the labeling of antibodies. This labeling density is equivalent to the labeling density in an observation image reconstructed from speckle images of 2×10³ frames in the IRIS method of the present inventors. As shown in the left view of FIG. 5 (d) and in FIG. 5 (e), actin filaments show a pattern that is not continuous in the longitudinal direction in an observation image reconstructed from speckle images of 2×10³ in the IRIS method. Even a labeling density that was 6.5 times its original was not sufficient for continuous staining of actin filaments (FIG. 5 (d)), however it was possible to achieve a labeling density of 1.2×10⁴ for an observation image reconstructed from speckle images of 2×10⁵ frames and to obtain consecutive super-resolution images of actin filaments (FIG. 5 (d) and FIG. 5 (e)). As described above, it was made obvious that the IRIS method of the present invention can eliminate the problem of a labeling density that had conventionally been an obstacle to dissolving two or three types of target substances coexisting close to each other.

For a cell that was fixed and had received a permeabilization process, in an observation image generated by using the IRIS method that utilizes a Lifeact probe of an actin filament, it was possible to dissolve two parallel actin bundles that were apart by 50 nm (FIG. 5 (d) through FIG. 5 (f)). By contrast, in an image resulting from adding SiMS images (speckle images) (which is equivalent to an image that can be obtained by a conventional immunofluorescence method), it was not possible to dissolve two such actin bundles (lower left view of FIG. 5 (g)). According to the IRIS method, it is possible to obtain consecutive observation images of an actin filament or a microtubule (below). This is remarkable progress from the conventional super-resolution microscopy (FIG. 10).

Establishment of Screening Method of IRIS Probe

The present inventors established an effective method for swiftly determining an IRIS probe for a different cell structure while taking into consideration a necessary molecular characteristic determined by data using a Lifeact probe. A probe candidate was generated by making a protein and EGFP fuse with each other, the protein and the EGFP being known to bind to a target substance. Live-cell fluorescence single molecule speckle (SiMS) microscopy (documents 11 and 12) is also effective for testing a probe candidate. However, the present inventors discovered that the binding specificity and dissociation dynamics of a probe candidate can easily be clarified by bringing crude lysate of a cell that expresses EGFP fused probe candidate into contact with a fixed cell. The inventors selected an IRIS probe in the following four steps:

(i) SiMS images (speckle images) of 10,000 frames were picked up at intervals of 50 ms or 100 ms, and probe candidates that were not able to localize in targets in an integrated SiMS image were excluded; (ii) probe candidates that were not able to be easily removed through washing were excluded; (iii) probe candidates having high dissociation speeds (with a half-life of 10 ms through 500 ms, see FIG. 11) were selected; and (iv) it was confirmed, in a reconstruction IRIS image (observation image), that probe candidates were able to localize in targets.

As shown in table 1, 18 types were selected by the above screening method from among the probe candidates. By a plurality of probes for microtubule and focal adhesion, different sites of their structure bodies were able to be visualized (FIG. 12 and FIG. 13). Also, independently from the above, it has been confirmed that Atto488-Lifeact is effective as a probe for an actin filament and is effective for the visualization of an actin filament (FIG. 9 etc.). This suggests that IRIS is effective for mapping a distribution of probe identification sites in one structure body.

Super-Resolution Image of a Plurality of Target Substances by IRIS

It was discovered that Lifeact, a CLIP-170 fragment (residues 3-309), a Plecin-1 fragment (residues 4022-4364) and a phosphatidyl inositol-(4)-phosphate 5-kinase type Iγ-90 (PIPKIγ) fragment (residues 641-668) are preferable respectively for observation of actin, microtubules, intermediate filaments and focal adhesion. By utilizing the exchangeability of IRIS probes, images in which the IRIS probes bound to 4 different types of cytoskeleton structures were obtained sequentially.

Further, the present inventors investigated a three-dimensional (3D) network of an actin filament, a microtubule and an intermediate filament. An image based on each IRIS probe was obtained by alternately using total internal reflection illumination and epi-illumination, and super-resolution images at the bottom and in the entire peripheral region of the cell were reconstructed respectively by using the obtained images. These images show that actin arcs running parallel to the cell contour gradually rise as if they were climbing on the radial actin bundles localizing at the bottom (arrows in FIG. 6 (a)) with decreasing distance to the center. It was confirmed that microtubules and intermediate filaments behind the lamellipodium (lobopodium) base were eliminated from the cell bottom at several locations (arrows in FIG. 6 (b) and FIG. 6 (c)).

The z position of each observation target object was estimated by using a signal ratio between an IRIS image based on TIRF and an IRIS image based on epi-fluorescence on the basis of measurements that used images of tilted fluorescent microtubules described in an above method (document 18). The 3D images as shown in FIG. 15 clarified the structure of the target substance. Lamellipodia (LP), stress fibers (SF) and actin arcs (Arc) were distributed with height positions of their centers of gravity (average ±S.D.) of 42±43 nm, 34±30 nm and 217±132 nm, respectively (arrows in FIG. 15 (a) and FIG. 15 (d)). The z positions of the microtubules became lower from 150 nm through 200 nm to 50 nm through 100 nm in the vicinity of the cell perimeters (arrowheads in FIG. 15 (b) and FIG. 15 (e)). Intermediate filaments formed mesh-shaped structures throughout the cell body, and some of the filaments were located at a height of approximately 200 nm behind the lamellipodium (ellipse in FIG. 15 (c)).

An IRIS image makes it possible to observe a spatial relationship between a plurality of cytoskeletal structures in a single cell at a resolution capability exceeding the diffraction limit. In a lamellar region, intermediate filaments were tangled with actin stress fibers in many cases, but they are not tangled with microtubules (FIG. 7 (a) through FIG. 7 (c)). The tangled actins and intermediate filaments appear to be linked to each other. Their cross-sectional profiles show that actin stress fibers overlapped intermediate filaments (arrows in FIG. 7 (e)). In peripheral regions, intermediate filaments did not tangle with actin stress fibers (FIG. 7 (g) and FIG. 7 (i)), whereas some intermediate filaments ran along microtubules (arrows in FIG. 7 (h)). The cross-sectional profiles show that intermediate filaments overlapped microtubules but did not overlap actin filaments (arrows in FIG. 3 (i), and FIG. 3 (j)). Thus, IRIS can reveal a region-specific proximity between 4 cytoskeletal components.

Behavior of Plus End of Microtubule in Live Cell and Comparison with Super-Resolution of Cytoskeleton Network

The present inventors discovered that the heights of microtubules locally change in the vicinity of focal adhesions and stress fibers. When microtubules crossed focal adhesions and stress fibers, they were lifted to positions at 200 nm from positions at 100 nm from the glass surface (arrowheads in FIG. 8 (a), FIG. 8 (b) and FIG. 16). Components of focal adhesions are located at a height of 30 nm through 80 nm from the glass surface (document 19). It appears that the lifted microtubules climbed on actin stress fibers without touching focal adhesions.

The present inventors further investigated a relationship between a microtubule plus end and a cytoskeleton network in that portion by observing the behavior of EB1-EGFP in a live cell and then reconstructing a super-resolution image of a 3D cytoskeleton network after fixation. The imaging system of the present inventors makes it possible to perform live-cell 3D imaging with acousto-optic tunable filters for swiftly switching between total internal reflection illumination and epi-illumination. The trajectory of the tip of the EB1-labeled microtubule was compared with focal adhesions and stress fibers in a super-resolution image (asterisk in FIG. 8 (c)). When the tip of the microtubule glowing was brought into contact with a stress fiber, the tip moved upward so as to move away from a focal adhesion existing below (FIG. 8 (d)). When the tip of the EB1-labeled microtubule was brought into contact with a stress fiber, the growth slowed down and the moving direction changed (arrow in FIG. 8 (d)). These pieces of data suggest that the speed and the direction of growth of a microtubule are greatly influenced by the collision and subsequent interaction with an actin stress fiber. As described above, a combination between IRIS and live-cell imaging makes it possible to clarify formation processes of a plurality of cytoskeletal structures that dynamically interact with each other.

Consideration

Consideration 1: Comparison with PAINT

Document 20 (Proceedings of the national Academy of Sciences of the United States of America 103, 18911-18916 (2006)) has reported a method called PAINT (point accumulation for imaging in nanoscale topography). This document has reported that super-resolution observation based on PAINT was conducted on lipid bilayer morphology by using Nile-red, which is a fluorescent dye that rapidly shuttles between an aqueous solution and the lipid bilayer. However, Nile-red is not a probe that binds to and dissociates from a specific bilayer molecule type. This prevents highly accurate determination of the position of a specific lipid molecule type that constitutes a lipid layer. By contrast, a probe of IRIS can bind to and dissociate from a specific target protein from among various types of proteins existing in a cell so as to make it possible to determine the position of the molecule thereof highly accurately. In addition, the concept of PAINT does not include a concept of obtaining a distribution of a plurality of target substances through probe exchange of IRIS. High resolution-capability imaging of various target substances by IRIS cannot be realized by the concept of PAINT. Also, this document has does not disclose improving of resolution by increasing the number of times of observation in PAINT. The reason appears to be that even an increased number of times of observation does not fix a lipid layer, which is a target substance, and thus the resolution does not improve through a change in the shape. In addition, the reason also seems to be that the problem of labeling density first became widely recognized a few years later than the document as an unsolved problem among those skilled in the art (documents 4 through 7).

The problem of labeling density was proposed in 2008 (document 6), whereas it had long been an unsolved problem in super-resolution microscope observation (documents 4, 5 and 7). As a method of increasing a target density, the review of 2013 (document 5) introduced an attempt to make fluorescent dye, which is an organic compound that is smaller than an antibody and a fluorescent protein, stably bind to a target substance and to make a single domain antibody such as Nanobody etc., which stably binds, stably bind to a target substance. This means that PAINT had not been recognized as a method of increasing a target density. Also, even the method introduced in the above review did not achieve a labeling density equivalent to that realized by the present invention, and the problem of a labeling density had remained unsolved. From these points, it can be said that there had not been an idea of using a binding and dissociation probe for solving the problem of a labeling density. In FIG. 10, an observation image of a microtubule based on IRIS and those based on STORM and Exchange-PAINT, which are conventionally known super-resolution microscopy, are compared (documents 21 to 23). According to the line profiles of label intensities along the longitudinal direction of microtubules, the observation image based on IRIS shows a more continuous pattern than those of observation images based on STORM and Exchange-PAINT (FIG. 10 (b)). Also, it is easy for the labeling density of actin filaments based on IRIS to become 60 times the maximum density of antibodies that bind to actin filaments. From these results, it is obvious that IRIS according to the present invention can solve the problem of labeling density that has caused a deterioration in reliability of the conventional super-resolution microscopy.

In addition to the ability to overcome the problem of labeling density, according to IRIS of the present invention, it is easy to visualize a plurality of target substances by protein-based exchangeable probes and there are no limitations on the number of target substances. In super-resolution microscopy that has conventionally been known and available, only up to 2 target substances can be observed (documents 21, 22, 24 and 25). Recently, Exchange-PAINT, which makes it possible to conduct super-resolution observation of a plurality of target substances, was announced (document 23 (Nature methods, 11, 313-318, 2014)). Exchange-PAINT uses characteristic labeling means of hybridizing short and fluorescence-labeled DNA with complementary DNA conjugated to antibody molecules. Exchange-PAINT is similar to IRIS in that it sequentially visualizes a plurality of target substances one by one by sequentially hybridizing pairs of various types of oligonucleotides. However, in Exchange-PAINT, uneven labeling and/or interference between antibodies sometimes lead to inaccurate analysis of the distribution of labeling substance (FIG. 10 (b)). By contrast, an IRIS probe is washed and removed and thereafter the next IRIS probe is brought into contact with the sample, resulting in no interference between a plurality of probes that are respectively for a plurality of target substances. The data of the present inventors shows the region-specific proximity of three types of cytoskeleton structures and focal adhesions with an accuracy exceeding the diffraction limit. In principle, the number of target substances that can be observed by IRIS is not limited even when they coalesce in a narrow region. This effect is remarkably advantageous in comparison with existing super-resolution microscopy.

Consideration 2: Probe

As shown in the above table, the present inventors have confirmed that probes of Document 19 are actually effective for the IRIS method. The above probes have been probes that use a binding partner known to associate with a target substance material, however the scope of the invention is not limited to this example. Using a phage display or an assay that confirms interactions between proteins of yeast-two hybrid system etc. makes it possible to perform screening for useful IRIS probes.

A plurality of IRIS probes for a microtubule and a focal adhesion visualized different sites of their structure bodies. A MAP4 fragment and a tau protein weakly visualized a microtubule plus end when EB1 existed (FIG. 12 (e)). A CLIP-170 fragment strongly labeled a microtubule tip when EB1 existed and continuously labeled the entire microtubule when EB1 did not exist (FIG. 12 (c) and FIG. 12 (e)). The result corresponds to in-vitro data (document 26). Further, Paxillin and the Src fragment visualized different portions of focal adhesion (FIG. 13). Thus, IRIS can also be applied to mapping analysis of a site in which a plurality of protein fragments bind. Development of an IRIS probe that binds to a molecule in a specific state also makes it is possible to perform super-resolution mapping on the molecule in the specific state.

2. Experiment 2

2.1. Method of Producing a Fab Probe from a Polyclonal Antibody

An anti-p40 antibody including antiserum was produced from a rabbit by using an antigen Xenopus laevis derived p40 produced in the present inventors' laboratory. Rabbit antiserum was put into an affinity column filled with antigens and polyclonal antibodies were made to adsorb on the column. pH in the column was reduced in a stepwise manner (pH5 to pH2) and the antibodies made to adsorb were eluted. 2 ml of fraction eluted at a high pH (pH2 through pH3.5) was added to 1.4 ml of a 50% slurry solution of Protein A Protein beads (Protein A Sepharose CL-4B, GE) and the antibodies were made to adsorb on the beads over 1 night at 4 degrees Celsius. After using PBS to wash and remove antibodies that had not been made to adsorb, the beads were suspended in 1 ml of PBS. To this suspension of beads, 50 μl of 1 μg/μl DyLight 488 NHS Ester (ThermoScientific) in which DMSO was dissolved was added, and antibodies were fluorescence-labeled at an ambient temperature over one hour. DyLight 488 NHS Ester that had made no reactions was washed and removed by using PBS and only beads were collected through centrifugation. In order to produce a Fab fragment from an antibody that was made to adsorb on beads, 1 ml of 7 μg/ml papain dissolved in a Digestion Buffer (50 mM Tris-HCl pH8.0, 10 mM Cysteine-HCl, 2 mM EDTA) was added and reactions were caused for 1 hour at 37 degrees Celsius. After the centrifugation, supernatant containing a Fab fragment was collected and 1 μl of 1 mg/ml leupeptin was added in order to inhibit the activity of papain. The obtained Fab fragment was put into an affinity column filled with antigens, and the Fab fragment was made to adsorb. pH in the column was reduced in a stepwise manner and a Fab fragment of a fraction eluted at a high pH (pH 5 to pH 4) was obtained. As described above, in the production of a Fab fragment from a polyclonal antibody, column purification was conducted twice. By using the affinity column of the first time, an antibody having a weak antigen-binding force was purified. From that antibody, a Fab fragment having a further weak antigen-binding force was produced. By using the affinity column of the second time, a Fab fragment in which an antigen-binding force still remained was purified. A Fab fragment prepared in this method was derived from IgG.

2.2. Iris Super-Resolution Imaging of Actin

Preparation of Arp2/3 complex observation cell sample By following the procedures described in detail in “procedures for imaging of multicolor super-resolution by IRIS” of experiment 1, a Xenopus laevis XTC cell was fixed and received a permeabilization process. After a blocking step with 4% bovine serum albumin for 30 minutes, the Fab probes were brought into contact with the cells in an imaging solution comprising the HEPES-buffered solution (10 mM Hepes pH 7.2, 90 mM KCl, 3 mM MgCl2, 100 μM DTT, 0.1% Triton X-100) supplemented with an oxygen-scavenging mix (200 μg/ml glucose oxidase, 35 μg/ml catalase, 4.5 mg/ml glucose, 0.5% 2-mercaptoethanol). The Fab probe concentration was 100 nM in the imaging solution.

Imaging and Image Reconstruction

Similarly to “procedures for imaging of multicolor super-resolution by IRIS” of experiment 1, SiMS images (speckle images) were obtained by using an inverted microscope (Olympus IX83-ZDC) equipped with an Olympus PlanApo 100×/1.45-numerical aperture (NA) objective lens, a 2×intermediate lens and an EM-CCD camera (Evolve 512, Roper) and controlled by MetaMorph software (Molecular Device). The Fab probe fluorescence-labeled by being irradiated with a 488 nm laser beam (50 mW) was excited for TIRF observation. SiMS images (speckle images) of a total of 33,750 frames were picked up by conducting consecutive imaging of 250 frames for each under a condition that the exposure time was 100 milliseconds for 1 frame and the frame rate (imaging speed) was 10 Hz (10 frames per second).

The procedures for image reconstruction from the above SiMS images of 33,750 frames are as described in detail in “procedures for image reconstruction in IRIS” of experiment 1.

The half-life of a probe-target complex formed between a purified Fab probe and a target antigen was obtained in a similar method to that in Experiment 1. In other words, by using the SiMS images picked up in the above, a period of time between when the Fab probe that had bound to the target appeared in a speckle image and it disappeared through dissociation was measured in a semi-manual mode by using Speckle TrackerJ, a plug-in of ImageJ. Then, the number of binding probes with respect to the period of time between the appearance and disappearance was plotted in accordance with a complementary cumulative relative frequency function (1-Ndissociation). Then, by fitting the complementary cumulative relative frequency function with an exponent function, the half-life was calculated. The result showed that the half-life of a probe-target complex formed between the Fab probe and a target antigen was 216 milliseconds.

2.3. Results

FIG. 17 shows a reconstruction image based on the Fab probe generated from a polyclonal antibody derived from a rabbit with respect to a p40 subunit of an Arp2/3 complex, which is an actin polymerization promotion factor. It is known that much p40 is distributed in a peripheral region of a cell and a distribution of Fab probes was visualized as such.

3. Experiment 3

Screening of Fab Probe

A library (1200 samples) of hybridoma was generated by using FLAG peptide (sequence number 2) as an antigen. After immunizing a mouse with an antigen, lymphocytes were collected from an iliac lymph node and were made to fuse with a mouse myeloma cell, and hybridoma was generated. The library of hybridoma was made through dilution culture. Culture supernatant was collected for each sample from the hybridoma library and was made to react with a 96 well glass bottom plate in which Protein G was solid phased and thereby an antibody in the culture supernatant was fixed to the surface of the bottom glass.

The 96 well glass bottom plate in which Protein G was solid phased and immobilization of the antibody were conducted in the following procedures. (3-aminopropyl) triethoxysilane was dissolved in a mixture of methanol and acetic acid (mixing ratio 100:5) and a 3% solution was produced. This solution was put in a 96 well multi-well plate having each of its wells made of glass, the solution was incubated for 30 minutes at an ambient temperature, and the glass surfaces of the well bottoms were coated. (3-aminopropyl) triethoxysilane that had made no reactions was washed a plurality of times with methanol so as to remove it and thereafter the plate was air dried so that (3-aminopropyl) triethoxysilane was made to remain on the glass surfaces. Next, a 0.1% glutaraldehyde solution was added to each well so as to cause reactions for 30 minutes at an ambient temperature and glutaraldehyde was made to bind to the glass surfaces. A 50 ng/μl Protein G (Thermo Scientific) solution was added to each well and was kept in contact with the glass surfaces to which glutaraldehyde bound, for 1 night at 4 degrees Celsius. Protein G causes covalent bonding with (3-aminopropyl) triethoxysilane via glutaraldehyde and is solid phased on the glass surfaces. Blocking was conducted on the glass surfaces on which Protein G was solid phased, with a 3% BSA solution for 1 night at 4 degrees Celsius. Thereafter, a hybridoma culture supernatant containing an antibody was added to each well and was left to stand for 1 night at 4 degrees Celsius. An antibody contained in the culture supernatant binds, specifically and strongly, to Protein G via the Fc domain thereof and is fixed to the glass surface. After removing the hybridoma culture supernatant, the glass surfaces were washed with a cell lysis buffer (10 mM Hepes pH 7.2, 3 mM MgCl2, 0.2% Triton X-100, 100 μM DTT), and received an observation step, which will be described later. In order to obtain a fused protein of FLAG peptide and FLAG peptide that becomes an antigen, an HEK-293F cell was transfected with a plasmid that encoded FLAG-EGFP. After culturing the transfected cell for 3 to 4 days, it was dissolved in a cell lysis buffer (10 mM Hepes pH 7.2, 3 mM MgCl2, 0.2% Triton X-100, 100 μM DTT) containing a protease inhibitor cocktail (Nacalai Tesque). After centrifugally separating the lysate, the supernatant liquid was collected. The FLAG-EGFP concentration contained in the supernatant liquid was estimated from the light emission intensity of EGFP, and a 50 nM FLAG-EGFP solution was prepared by diluting it with a cell lysis buffer.

A 50 nM FLAG-EGFP solution was added to each well to which an antibody was fixed. By using a TIRF microscope, SiMS images (speckle images) of 500 frames were picked up with an exposure time of 50 ms for 1 frame and at a frame rate of 20 Hz (20 frames per second). As a result of observing 1200 wells, 10 to 300 speckles (0.006 to 0.178 speckles/μm2) were observed in the field of view of the microscope (41 μm×41 μm). Each speckle corresponds to EGFP of a single molecule. As examples of speckle images, FIG. 18A shows an image in which about 250 speckles were observed and FIG. 18B shows an image in which about 20 speckles were observed. In the case where 50 nM FLAG-EGFP solutions were added to wells in which antibodies had not been solid phased, the numbers of the speckles in the field of view were 10 to 20 (0.006 to 0.012/μm2). Then, the half-lives of binding between antibodies and FLAG-EGFP were measured by treating, as positive examples, a 26 well in which 40 (0.023/μm2) or more speckles had been observed in the field of view. Each of the wells had different half-lives. The half-life in the well shown in FIG. 18A was 55 ms.

From the 26 well in which 40 or more speckles had been observed in the field of view of the microscope as the positive examples, antibodies having half-lives of the probe-target complex that were equal to or more than 10 milliseconds and equal to or less than 3 seconds were selected. Hybridoma producing these antibodies was converted into monoclone and 2 ml to 10 ml of the culture supernatant was collected. 40 μl of a 50% slurry solution of Protein A beads (Protein A Sepharose CL-4B, GE) was added to the culture superresolution, and the antibodies were made to adsorb on the beads for 1 night at 4 degrees Celsius. After using PBS to wash and remove antibodies that had not adsorbed, the beads were suspended in 100 μl of PBS. To this suspension of beads, 16 μl of 0.5 μg/μl DyLight 488 NHS Ester (ThermoScientific) dissolved in DMSO was added, and the antibodies were fluorescence-labeled for 1 hour at an ambient temperature. DyLight 488 NHS Ester that had made no reactions was washed and removed with PBS and only beads were collected after centrifugation. In order to produce a Fab fragment from an antibody that bound to the beads, 20 μl of 0.01 mg/ml papain dissolved in Digestion Buffer (50 mM Tris-HCl pH8.0, 10 mM Cysteine-HCl, 2 mM EDTA) was added and reactions were caused for 1 hour at 37 degrees Celsius. After the centrifugation, supernatant containing a Fab fragment was collected and 2 μl of 0.01 mg/ml leupeptin was added in order to inhibit the activity of papain.

2.2. IRIS Super-Resolution Imaging of Actin

Preparation of a FLAG Tag Fused Actin Expression XTC Cell Sample

FLAG tag fused actin encoded by the base sequence described by sequence number 20 was expressed with an XTC cell.

Expression vector-(CLONETECH) encoding pEGFP-actin was cut with restriction enzyme Age-I, Bgl-II (NEB) to remove EGFP from the vector. Synthetic cDNA encoding FLAG peptide (DYKDDDDK) was inserted into the vector and an expression plasmid encoding an actin for which FLAG was tagged to an N terminus was constructed.

A Xenopus laevis XTC cell was transfected with the above expression plasmid in the following procedures. 3 μl of an expression plasmid of 1 μg/μ of FLAG fused actin was dissolved in 200 μl of a serum-free medium (70% dilution Leibovitz's L-15 medium) and 8μ of Polyethylenimine, linear, M.W. 25,000 (Polysciences) of 1 mg/ml was added and it was vortexed. After leaving it to stand for 30 minutes at an ambient temperature, a medium with serum of 1 ml (70% dilution Leibovitz's L-15 medium, 10% FCS supplement) was added. The solution containing this plasmid and the media of XTC cells spread in 6 wells were switched, and the cells were made to take in plasmids for 1 night.

3 to 4 days later than the transfecting, the cells were fixed and received the permeabilization process in the procedures described in detail in “procedures for imaging of multicolor super-resolution by IRIS” of experiment 1. After a blocking step with 4% bovine serum albumin for 30 minutes, the Fab probes were each brought into contact with the cells in an imaging solution comprising the HEPES-buffered solution (10 mM Hepes pH 7.2, 3 mM MgCl2, 100 μM DTT, 1 μg/ml leupeptin) supplemented with an oxygen-scavenging mix (200 μg/ml glucose oxidase, 35 μg/ml catalase, 4.5 mg/ml glucose, 0.5% 2-mercaptoethanol). The Fab probe concentration was 3 nM in the imaging solution.

Imaging and Image Reconstruction

Similarly to “procedures for imaging of multicolor super-resolution by IRIS” of experiment 1, SiMS images (speckle images) were obtained by using an inverted microscope (Olympus IX83-ZDC) equipped with an Olympus PlanApo 100×/1.45-numerical aperture (NA) objective lens, a 2×intermediate lens and an electron-multiplying EM-CCD camera (Evolve 512, Roper) and controlled by MetaMorph software (Molecular Device). The Fab probe fluorescence-labeled by being irradiated with a 488 nm laser beam (50 mW) was excited for TIRF observation. SiMS images (speckle images) of a total of 120,000 frames were picked up by conducting consecutive imaging of 500 frames for each under a condition such that the exposure time was 50 milliseconds for 1 frame and the frame rate (imaging speed) was 20 Hz (20 frames per second).

The procedures for image reconstruction from the above SiMS images of 120,000 frames are as described in detail in “procedures for image reconstruction in IRIS” of experiment 1.

2.4. Results

FIG. 19 shows IRIS super-resolution images of FLAG fused actin by a Fab probe derived from an anti-FLAG monoclonal antibody selected by the above screening method. The half-life of a probe-target complex of a Fab probe and an FLAG fused actin used for the generation of these IRIS super-resolution images was 203 milliseconds.

The present screening method made it possible to select a Fab probe appropriate for IRIS super-resolution imaging.

4. Documents

The documents referred to herein are described below.

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The present invention can provide a super-resolution microscope observation method that makes it possible to obtain, at a high density, position information of luminescent substances used for labeling and to generate a high resolution observation image exceeding the diffraction limit, and also it is made possible to clarify the formation process of a plurality of cytoskeleton structures that interact dynamically, by a combination between the present invention and a living-cell image technique, and thus the present invention has a high industrial applicability.

All publications, patents and patent applications referred to herein are incorporated herein in their entirety by reference. 

What is claimed is:
 1. An observation method of a sample containing a target substance, the observation method comprising: an imaging step in which a step of obtaining a speckle image including, as a speckle, light emitted from a luminescent substance under a prescribed condition in a state in which a medium is brought into contact with the sample is performed a plurality of times at different times respectively so as to obtain a plurality of speckle images, the medium containing a probe that contains the luminescent substance emitting light under the prescribed condition and that repeatedly binds to and dissociates from the target substance directly and specifically; and an observation image generation step of generating an observation image of the target substance in the sample from the plurality of speckle images, wherein a half-life of a probe-target complex formed by binding between the probe and the target substance is equal to or more than 10 milliseconds and equal to or less than 3 seconds.
 2. The method according to claim 1, wherein the observation image generation step is a step in which information of a position of a speckle included in each of the plurality of speckle images is obtained for each of the plurality of speckle images and the observation image is generated on the basis of the information from the plurality of speckle images.
 3. The method according to claim 1, wherein the sample includes two or more target substances, the imaging step is sequentially performed on the sample by using the probe that is specific to each of the target substances, and the observation image generation step is a step in which observation images of the respective target substances in the sample are respectively generated from the plurality of speckle images obtained from the respective imaging steps.
 4. The method according to claim 3, further comprising a multiple-observation image generation step in which observation images of the respective target substances in the sample generated in the observation image generation step are superposed so as to generate a multiple-observation image, which is an observation image of the two or more target substances in the sample.
 5. The method according to claim 1, wherein the luminescent substance is a fluorescent substance, and the prescribed condition is irradiation with excitation light.
 6. The method according to claim 1, wherein a combination between the probe and the target substance is selected from a group of: a combination wherein the probe is (a1) a polypeptide, linked to the luminescent substance, which consists of an amino acid sequence of sequence number 19, (a2) a polypeptide, linked to the luminescent substance, which consists of the amino acid sequence described in (a1) where one or a plurality of amino acids have been substituted, deleted, inserted or added and for which a half-life of a probe-target complex formed by binding between the probe and the target substance is equal to or more than 10 milliseconds and equal to or less than 3 seconds, or (a3) a polypeptide, linked to the luminescent substance, which consists of an amino acid sequence having at least a 70% identity with the amino acid sequence described in (a1) and for which a half-life of a probe-target complex formed by binding between the probe and the target substance is equal to or more than 10 milliseconds and equal to or less than 3 seconds, and the target substance is an actin polymer; a combination wherein the probe is (b1) a polypeptide, linked to the luminescent substance, which consists of an amino acid sequence that is a partial amino acid sequence of an amino acid sequence of sequence number 12, that at least partially contains an amino acid sequence of 3-309 and that has 407 or fewer amino acids, an amino acid sequence that is a partial amino acid sequence of an amino acid sequence of sequence number 14, that at least partially contains an amino acid sequence of 2536-2843 and that has 408 or fewer amino acids, an amino acid sequence that is a partial amino acid sequence of an amino acid sequence of sequence number 14, that at least partially contains an amino acid sequence of 2781-2819 and that has 138 or fewer amino acids, an amino acid sequence that is a partial amino acid sequence of an amino acid sequence of sequence number 4, that at least partially contains an amino acid sequence of 1-908 and that has 1008 or fewer amino acids, an amino acid sequence that is a partial amino acid sequence of an amino acid sequence of sequence number 4, that at least partially contains an amino acid sequence of 659-908 and that has 394 or fewer amino acids, an amino acid sequence of sequence number 5, or an amino acid sequence of sequence number 6, (b2) a polypeptide, linked to the luminescent substance, which consists of the amino acid sequence described in (b1) where one or a plurality of amino acids have been substituted, deleted, inserted or added and for which a half-life of a probe-target complex formed by binding between the probe and the target substance is equal to or more than 10 milliseconds and equal to or less than 3 seconds, or (b3) a polypeptide, linked to the luminescent substance, which consists of an amino acid sequence having at least a 70% identity with the amino acid sequence described in (b1) and for which a half-life of a probe-target complex formed by binding between the probe and the target substance is equal to or more than 10 milliseconds and equal to or less than 3 seconds, and the target substance is a microtubule; a combination wherein the probe is (c1) a polypeptide, linked to the luminescent substance, which consists of an amino acid sequence that is a partial amino acid sequence of an amino acid sequence of sequence number 8, that at least partially contains an amino acid sequence of 3777-4684 and that has 1008 or fewer amino acids, an amino acid sequence that is a partial amino acid sequence of an amino acid sequence of sequence number 8, that at least partially contains an amino acid sequence of 3777-4364 and that has 688 or fewer amino acids, an amino acid sequence that is a partial amino acid sequence of an amino acid sequence of sequence number 8, that at least partially contains an amino acid sequence of 3777-4313 and that has 637 or fewer amino acids, or an amino acid sequence that is a partial amino acid sequence of an amino acid sequence of sequence number 8, that at least partially contains an amino acid sequence of 4022-4364 and that has 443 or fewer amino acids, (c2) a polypeptide, linked to the luminescent substance, which consists of the amino acid sequence described in (c1) where one or a plurality of amino acids have been substituted, deleted, inserted or added and for which a half-life of a probe-target complex formed by binding between the probe and the target substance is equal to or more than 10 milliseconds and equal to or less than 3 seconds, or (c3) a polypeptide, linked to the luminescent substance, which consists of an amino acid sequence having at least a 70% identity with the amino acid sequence described in (c1) and for which a half-life of a probe-target complex formed by binding between the probe and the target substance is equal to or more than 10 milliseconds and equal to or less than 3 seconds, and the target substance is an intermediate filament; and a combination wherein the probe is (d1) a polypeptide, linked to the luminescent substance, which consists of an amino acid sequence of sequence number 15, an amino acid sequence that is a partial amino acid sequence of an amino acid sequence of sequence number 15, that at least partially contains an amino acid sequence of 54-557 and that has 556 or fewer amino acids, an amino acid sequence that is a partial amino acid sequence of an amino acid sequence of sequence number 15, that at least partially contains an amino acid sequence of 54-498 and that has 545 or fewer amino acids, an amino acid sequence that is a partial amino acid sequence of an amino acid sequence of sequence number 15, that at least partially contains an amino acid sequence of 167-557 and that has 491 or fewer amino acids, an amino acid sequence that is a partial amino acid sequence of an amino acid sequence of sequence number 16, that at least partially contains an amino acid sequence of 1-251 and that has 351 or fewer amino acids, an amino acid sequence that is a partial amino acid sequence of an amino acid sequence of sequence number 16, that at least partially contains an amino acid sequence of 3-251 and that has 349 or fewer amino acids or an amino acid sequence of sequence number 18, (d2) a polypeptide, linked to the luminescent substance, which consists of the amino acid sequence described in (d1) where one or a plurality of amino acids have been substituted, deleted, inserted or added and for which a half-life of a probe-target complex formed by binding between the probe and the target substance is equal to or more than 10 milliseconds and equal to or less than 3 seconds, or (d3) a polypeptide, linked to the luminescent substance, which consists of an amino acid sequence having at least a 70% identity with the amino acid sequence described in (d1) and for which a half-life of a probe-target complex formed by binding between the probe and the target substance is equal to or more than 10 milliseconds and equal to or less than 3 seconds, and the target substance is a focal adhesion.
 7. The method according to claim 1, wherein the probe contains an antibody or a fragment of an antibody, the antibody or the fragment being to the target substance and the antibody or the fragment being linked to the luminescent substance.
 8. The method according to claim 7, wherein the fragment of the antibody is a Fab fragment.
 9. A probe used for labeling a target substance, wherein the probe contains a luminescent substance that emits light under a prescribed condition, the probe can repeatedly bind to and dissociate from the target substance directly and specifically, and a half-life of a probe-target complex formed by binding to the target substance is equal to or more than 10 milliseconds and equal to or less than 3 seconds.
 10. The probe according to claim 9, wherein the target substance is an actin polymer and the probe is (a1) a polypeptide, linked to the luminescent substance, which consists of an amino acid sequence of sequence number 19, (a2) a polypeptide, linked to the luminescent substance, which consists of the amino acid sequence described in (a1) where one or a plurality of amino acids have been substituted, deleted, inserted or added and for which a half-life of a probe-target complex formed by binding between the probe and the target substance is equal to or more than 10 milliseconds and equal to or less than 3 seconds, or (a3) a polypeptide, linked to the luminescent substance, which consists of an amino acid sequence having at least a 70% identity with the amino acid sequence described in (a1) and for which a half-life of a probe-target complex formed by binding between the probe and the target substance is equal to or more than 10 milliseconds and equal to or less than 3 seconds, the target substance is a microtubule and the probe is (b1) a polypeptide, linked to the luminescent substance, which consists of an amino acid sequence that is a partial amino acid sequence of an amino acid sequence of sequence number 12, that at least partially contains an amino acid sequence of 3-309 and that has 407 or fewer amino acids, an amino acid sequence that is a partial amino acid sequence of an amino acid sequence of sequence number 14, that at least partially contains an amino acid sequence of 2536-2843 and that has 408 or fewer amino acids, an amino acid sequence that is a partial amino acid sequence of an amino acid sequence of sequence number 14, that at least partially contains an amino acid sequence of 2781-2819 and that has 138 or fewer amino acids, an amino acid sequence that is a partial amino acid sequence of an amino acid sequence of sequence number 4, that at least partially contains an amino acid sequence of 1-908 and that has 1008 or fewer amino acids, an amino acid sequence that is a partial amino acid sequence of an amino acid sequence of sequence number 4, that at least partially contains an amino acid sequence of 659-908 and that has 394 or fewer amino acids, an amino acid sequence of sequence number 5 or an amino acid sequence of sequence number 6, (b2) a polypeptide, linked to the luminescent substance, which consists of the amino acid sequence described in (b1) where one or a plurality of amino acids have been substituted, deleted, inserted or added and for which a half-life of a probe-target complex formed by binding between the probe and the target substance is equal to or more than 10 milliseconds and equal to or less than 3 seconds, or (b3) a polypeptide, linked to the luminescent substance, which consists of an amino acid sequence having at least a 70% identity with the amino acid sequence described in (b1) and for which a half-life of a probe-target complex formed by binding between the probe and the target substance is equal to or more than 10 milliseconds and equal to or less than 3 seconds, the target substance is an intermediate filament and the probe is (c1) a polypeptide, linked to the luminescent substance, which consists of an amino acid sequence that is a partial amino acid sequence of an amino acid sequence of sequence number 8, that at least partially contains an amino acid sequence of 3777-4684 and that has 1008 or fewer amino acids, an amino acid sequence that is a partial amino acid sequence of an amino acid sequence of sequence number 8, that at least partially contains an amino acid sequence of 3777-4364 and that has 688 or fewer amino acids, an amino acid sequence that is a partial amino acid sequence of an amino acid sequence of sequence number 8, that at least partially contains an amino acid sequence of 3777-4313 and that has 637 or fewer amino acids or an amino acid sequence that is a partial amino acid sequence of an amino acid sequence of sequence number 8, that at least partially contains an amino acid sequence of 4022-4364 and that has 443 or fewer amino acids, (c2) a polypeptide, linked to the luminescent substance, which consists of the amino acid sequence described in (c1) where one or a plurality of amino acids have been substituted, deleted, inserted or added and for which a half-life of a probe-target complex formed by binding between the probe and the target substance is equal to or more than 10 milliseconds and equal to or less than 3 seconds, or (c3) a polypeptide, linked to the luminescent substance, which consists of an amino acid sequence having at least a 70% identity with the amino acid sequence described in (c1) and for which a half-life of a probe-target complex formed by binding between the probe and the target substance is equal to or more than 10 milliseconds and equal to or less than 3 seconds, or the target substance is a focal adhesion and the probe is (d1) a polypeptide, linked to the luminescent substance, which consists of an amino acid sequence of sequence number 15, an amino acid sequence that is a partial amino acid sequence of an amino acid sequence of sequence number 15, that at least partially contains an amino acid sequence of 54-557 and that has 556 or fewer amino acids, an amino acid sequence that is a partial amino acid sequence of an amino acid sequence of sequence number 15, that at least partially contains an amino acid sequence of 54-498 and that has 545 or fewer amino acids, an amino acid sequence that is a partial amino acid sequence of an amino acid sequence of sequence number 15, that at least partially contains an amino acid sequence of 167-557 and that has 491 or fewer amino acids, an amino acid sequence that is a partial amino acid sequence of an amino acid sequence of sequence number 16, that at least partially contains an amino acid sequence of 1-251 and that has 351 or fewer amino acids, an amino acid sequence that is a partial amino acid sequence of an amino acid sequence of sequence number 16, that at least partially contains an amino acid sequence of 3-251 and that has 349 or fewer amino acids, or an amino acid sequence of sequence number 18, (d2) a polypeptide, linked to the luminescent substance, which consists of the amino acid sequence described in (d1) where one or a plurality of amino acids have been substituted, deleted, inserted or added and for which a half-life of a probe-target complex formed by binding between the probe and the target substance is equal to or more than 10 milliseconds and equal to or less than 3 seconds, or (d3) a polypeptide, linked to the luminescent substance, which consists of an amino acid sequence having at least a 70% identity with the amino acid sequence described in (d1) and for which a half-life of a probe-target complex formed by binding between the probe and the target substance is equal to or more than 10 milliseconds and equal to or less than 3 seconds.
 11. The probe according to claim 9, wherein the probe contains an antibody or a fragment of an antibody, the antibody or the fragment being to the target substance and the antibody or the fragment being linked to the luminescent substance.
 12. The probe according to claim 11, wherein the fragment of the antibody is a Fab fragment.
 13. A reagent kit for labeling a target substance, wherein the reagent kit at least includes the probe according to claim
 9. 14. A screening method of a site which identifies a target substance in the probe according to claim 9, the screening method comprising: an immobilization step in which a candidate substance of the site or a substance partially containing the candidate substance is fixed to a solid support; an observation step in which a target substance linked to a luminescent substance and a solid support obtained in the immobilization step are observed in a medium while the target substance linked to a luminescent substance and the solid support obtained in the immobilization step are kept in contact, in a condition that allows observation, in units of 1 molecule, of light emission from the luminescent substance in a probe-target complex formed by binding between the target substance and the candidate substance, and a screening step in which the candidate substance resulting in a half-life of the probe-target complex that is equal to or more than 10 milliseconds and equal to or less than 3 seconds is selected as the site on the basis of observation in the observation step.
 15. The method according to claim 14 wherein the candidate substance is an antibody or a fragment of an antibody from a library of hybridoma that produces an antibody to the target substance, and the antibody is fixed to a solid support in the immobilization step. 